Retentate chromatography and protein chip arrays with applications in biology and medicine

ABSTRACT

This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of the priority dates ofco-pending application Ser. No. 60/054,333 filed Jun. 20, 1997 andco-pending application Ser. No. 60/067,484 filed Dec. 1, 1997, thecontents of which are incorporated herein by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] This invention relates to the field of separation science andanalytical biochemistry.

[0004] The methods of this invention have applications in biology andmedicine, including analysis of gene function, differential geneexpression, protein discovery, cellular and clinical diagnostics anddrug screening.

[0005] Cell function, both normal and pathologic, depends, in part, onthe genes expressed by the cell (i.e., gene function). Gene expressionhas both qualitative and quantitative aspects. That is, cells may differboth in terms of the particular genes expressed and in terms of relativelevel of expression of the same gene. Differential gene expression canbe manifested, for example, by differences in the expression of proteinsencoded by the gene, or in post-translational modifications of expressedproteins. For example, proteins can be decorated with carbohydrates orphosphate groups, or they can be processed through peptide cleavage.Thus, at the biochemical level, a cell represents a complex mixture oforganic biomolecules.

[0006] One goal of functional genomics (“proteomics”) is theidentification and characterization of organic biomolecules that aredifferentially expressed between cell types. By comparing expression onecan identify molecules that may be responsible for a particularpathologic activity of a cell. For example, identifying a protein thatis expressed in cancer cells but not in normal cells is useful fordiagnosis and, ultimately, for drug discovery and treatment of thepathology. Upon completion of the Human Genome Project, all the humangenes will have been cloned, sequenced and organized in databases. Inthis “post-genome” world, the ability to identify differentiallyexpressed proteins will lead, in turn, to the identification of thegenes that encode them. Thus, the power of genetics can be brought tobear on problems of cell function.

[0007] Differential chemical analyses of gene expression and functionrequire tools that can resolve the complex mixture of molecules in acell, quantify them and identify them, even when present in traceamounts. However, the current tools of analytical chemistry for thispurpose are limited in each of these areas. One popular biomolecularseparation method is gel electrophoresis. Frequently, a first separationof proteins by isoelectric focusing in a gel is coupled with a secondseparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The result is a map that resolves proteins according to thedimensions of isoelectric point (net charge) and size (i.e., mass).However useful, this method is limited in several ways. First, themethod provides information only about two characteristics of abiomolecule—mass and isoelectric point (“pI”). Second, the resolutionpower in each of the dimensions is limited by the resolving power of thegel. For example, molecules whose mass differ by less than about 5% orless than about 0.5 pI are often difficult to resolve. Third, gels havelimited loading capacity, and thus sensitivity; one may not be able todetect biomolecules that are expressed in small quantities. Fourth,small proteins and peptides with a molecular mass below about 10-20 kDaare not observed.

[0008] Other analytical methods may overcome one or more of theselimitations, but they are difficult to combine efficiently. For example,analytical chromatography can separate biomolecules based on a varietyof analyte/adsorbent interactions, but multi-dimensional analysis isdifficult and time consuming. Furthermore, the methods are limited insensitivity.

[0009] Clinical diagnostics requires the ability to specifically detectknown markers of disease. However, the development of such diagnosticsis hampered by the time necessary to prepare reagents that specificallybind to markers, or that can discriminate the marker in a complexmixture.

[0010] Drug discovery requires the ability to rapidly screen agents thatmodulate ligand/receptor interactions. Often the rate-limiting step insuch screens is the ability to detect the ligand/receptor interaction.Thus, rapid and specific methods for identifying binding events would bean advance in the art.

[0011] Until now, the process from identifying a potential marker ormember of a ligand/receptor pair to producing an agent that specificallybinds the marker or member has been difficult. In one method, normal anddiseased tissue are compared to identify mRNA species or expressedsequence tags (“ESTs”) that are elevated or decreased in the diseasedtissue. These species are isolated and the polypeptides they encode areproduced through routine methods of recombinant DNA. Then, thepolypeptides are isolated and used as immunogens to raise antibodiesspecific for the marker. The antibodies can be used in, for example,ELISA assays to determine the amount of the marker in a patient sample.

[0012] This process is long and tedious. It can take nine months to ayear to produce such antibodies, with much of the time being spent ondeveloping protocols to isolate a sufficient quantity of the polypeptidefor immunization. Furthermore, the method relies on the hope thatdifferences in RNA expression are expressed as differences in proteinexpression. However, this assumption is not always reliable. Therefore,methods in which differentially expressed proteins are detected directlyand in which specific ligands could be generated in significantlyshorter time would be of great benefit to the field.

[0013] Thus, tools for resolving complex mixtures of organicbiomolecules, identifying individual biomolecules in the mixture andidentifying specific molecular recognition events involving one or moretarget analytes are desirable for analytical biochemistry, biology andmedicine.

SUMMARY OF THE INVENTION

[0014] This invention provides devices and methods for retentatechromatography. Retentate chromatography is a combinatorial method toprovide high information resolution of analytes in complex mixturesthrough the use of multi-dimensional separation methods. It provides aunified analyte detection and functional analysis capability for biologyand medicine that is characterized by a single, integrated operatingsystem for the direct detection of analyte expression patternsassociated with gene function, protein function, cell function, and thefunction of whole organisms. In one aspect, this invention provides aunified operating system for the discovery or diagnosis of genefunction, protein function, or the function of entire macromolecularassemblies, cells, and whole organisms.

[0015] More particularly, analytes can be resolved in a variety oftwo-dimensional formats, thereby providing multi-dimensionalinformation. Analytes are first separated in at least two differentfirst dimensions based on their ability to be adsorbed to a stationaryphase under at least two different selectivity conditions, such asanionic/cationic potential, hydrophobicity/hydrophilicity, or specificbiomolecular recognition. Then the analytes are separated in a seconddimension based on mass by desorption spectrometry (e.g., laserdesorption mass spectrometry), which further provides detection of theseparated analytes. The nature of the adsorbent to which the analytesadsorb provides physico-chemical information about the analyte.

[0016] Thus, this invention provides a molecular discovery anddiagnostic device that is characterized by the inclusion of bothparallel and multiplex analyte processing capabilities. Because analytesare directly detected, the invention enables the simultaneoustransmission of two or more independent target analyte signals from thesame “circuit” (i.e., addressable “chip” location) during a single unitoperation.

[0017] Retentate chromatography is distinct from conventionalchromatography in several ways. First, in retentate chromatography,analytes which are retained on the adsorbent are detected. Inconventional chromatographic methods analytes are eluted off of theadsorbent prior to detection. There is no routine or convenient meansfor detecting analyte which is not eluted off the adsorbent inconventional chromatography. Thus, retentate chromatography providesdirect information about chemical or structural characteristics of theretained analytes. Second, the coupling of adsorption chromatographywith detection by desorption spectrometry provides extraordinarysensitivity, in the femtomolar range, and unusually fine resolution.Third, in part because it allows direct detection of analytes, retentatechromatography provides the ability to rapidly analyze retentates with avariety of different selectivity conditions, thus providing rapid,multi-dimensional characterization of analytes in a sample. Fourth,adsorbents can be attached to a substrate in an array of pre-determined,addressable locations. This allows parallel processing of analytesexposed to different adsorbent sites (i.e., “affinity sites” or “spots”)on the array under different elution conditions.

[0018] Retentate chromatography has many uses in biology and medicine.These uses include combinatorial biochemical separation and purificationof analytes, the study of differential gene expression and molecularrecognition events, diagnostics and drug discovery.

[0019] One basic use of retentate chromatography as an analytical toolinvolves exposing a sample to a combinatorial assortment of differentadsorbent/eluant combinations and detecting the behavior of the analyteunder the different conditions. This both purifies the analyte andidentifies conditions useful for detecting the analyte in a sample.Substrates having adsorbents identified in this way can be used asspecific detectors of the analyte or analytes. In a progressiveextraction method, a sample is exposed to a first adsorbent/eluantcombination and the wash, depleted of analytes that are adsorbed by thefirst adsorbent, is exposed to a second adsorbent to deplete it of otheranalytes. Selectivity conditions identified to retain analytes also canbe used in preparative purification procedures in which an impure samplecontaining an analyte is exposed, sequentially, to adsorbents thatretain it, impurities are removed, and the retained analyte is collectedfrom the adsorbent for a subsequent round.

[0020] One aspect of the invention is that each class or type ofmolecular recognition event (e.g., target adsorbent-target analyteinteraction), characterized by a particular selectivity condition at anaddressable location within the array, is detected directly while theassociated molecules are still localized (i.e., “retained”) at theaddressable location. That is, selection and detection, by direct means,does not require elution, recovery, amplification, or labeling of thetarget analyte.

[0021] Another aspect of the present invention is that the detection ofone or more desirable molecular recognition events, at one or morelocations within the addressable array, does not require removal orconsumption of more than a small fraction of the totaladsorbent-analyte. Thus, the unused portion can be interrogated furtherafter one or more “secondary processing” events conducted directly insitu (i.e., within the boundary of the addressable location) for thepurpose of structure and function elucidation, including furtherassembly or disassembly, modification, or amplification (directly orindirectly).

[0022] Adsorbents with improved specificity for an analyte can bedeveloped by an iterative process, referred to as “progressiveresolution,” in which adsorbents or eluants proven to retain an analyteare tested with additional variables to identity combinations withbetter binding characteristics. Another method allows the rapid creationof substrates with antibody adsorbents specific for an analyte. Themethod involves docking the analyte to an adsorbent, and screening phagedisplay libraries for phage that bind the analyte.

[0023] Retentate chromatography has uses in molecular and cellularbiology, as well. Analytes that are differentially present in twosamples (e.g., differentially expressed proteins in two cell extracts)can be identified by exposing the samples to a variety ofadsorbent/eluant combinations for analysis by desorption spectrometry,thereby making use of the high information resolving power of the systemthat other separation and detections systems cannot match. Unknowntarget proteins can be identified by determining physicochemicalcharacteristics, including molecular mass, based on the chemicalcharacteristics of the adsorbent/eluant combination, and thisinformation can be used to screen databases for proteins having similarprofiles.

[0024] The methods in separation biochemistry and the adsorbentsproduced from these methods, are useful in diagnostics. Moreparticularly, adsorbents, either chemical or biospecific, can bedeveloped to detect important diagnostic markers. In certainembodiments, a substrate can have an array of adsorbent spots selectedfor a combination of markers diagnostic for a disease or syndrome.

[0025] Retentate chromatography also is useful in drug discovery. Onemember of a receptor/ligand pair is docked to an adsorbent, and itsability to bind the binding partner is tested in the presence of theagent. Because of the rapidity with which adsorption can be tested,combinatorial libraries of agents can be easily tested for their abilityto modulate the interaction.

[0026] In one aspect this invention provides a method for highinformation resolution of at least one analyte in a sample. The methodis a combinatorial separation method that includes separation anddetection of multiple analytes in parallel. The method comprises thesteps of a) exposing the analyte to at least two different selectivityconditions, each selectivity condition defined by the combination of anadsorbent and an eluant, to allow retention of the analyte by theadsorbent; and b) detecting retained analyte under the differentselectivity conditions by desorption spectrometry. Detection of retainedanalyte under the different selectivity conditions provides a highinformation resolution of the analyte.

[0027] In one embodiment each different selectivity condition is definedat a different predetermined, addressable location for parallelprocessing. In another embodiment, the method comprises the steps of i)exposing the analyte to a first selectivity condition at a definedlocation to allow retention of the analyte by the adsorbent; ii)detecting retained analyte under the first selectivity condition bydesorption spectrometry; iii) washing the adsorbent under a second,different selectivity condition at the defined location to allowretention of the analyte to the adsorbent; and iv) detecting retainedanalyte under the second selectivity condition by desorptionspectrometry.

[0028] In another embodiment the analyte is an organic biomolecule, amultimeric molecular complex or macromolecular assembly. In anotherembodiment the organic biomolecule is an enzyme, an immunoglobulin, acell surface receptor or an intracellular receptor.

[0029] In another embodiment the adsorbent comprises an anion, a cation,a hydrophobic interaction adsorbent, a polypeptide, a nucleic acid, acarbohydrate, a lectin, a dye, a reducing agent, a hydrocarbon or acombination thereof. In another embodiment the adsorbent is attached toa substrate comprising glass, ceramic, a magnetic material, an organicpolymer, a conducting polymer, a native biopolymer, a metal or a metalcoated with an organic polymer. In another embodiment the adsorbent isin the form of a microemulsion, a latex, a layer or a bead. In anotherembodiment the locations on the substrate are arranged in a line or anorthogonal array. In another embodiment the adsorbents are located on asubstrate at different locations before the analytes are exposed to theselectivity conditions. In another embodiment the adsorbents are locatedon a substrate at different locations after the analytes are exposed tothe selectivity conditions. In another embodiment the differentselectivity conditions comprise different binding conditions ordifferent elution conditions.

[0030] In another embodiment the step of detecting comprises detectingthe mass of the analyte by laser desorption mass spectrometry.

[0031] In another embodiment the selectivity conditions are selected tooptimize retention of analyte by an adsorbent. In another embodiment theat least one analyte is more than one analyte. In another embodiment theplurality of selectivity conditions are defined by at differentadsorbents and the same eluant.

[0032] Another embodiment further comprises the step of providing asubstrate comprising adsorbents at addressable locations, each adsorbentbeing an adsorbent from a selectivity condition identified to retain theanalyte. In another embodiment the elution conditions differ accordingto pH, buffering capacity, ionic strength, a water structurecharacteristic, detergent type, detergent strength, hydrophobicity ordielectric constant. In another embodiment the plurality of selectivityconditions are defined by the same eluant.

[0033] In another embodiment this invention provides a method forsequential extraction of analytes from a sample. This is acombinatorial, serial separation and purification development method formultiple analytes in parallel. The method comprises the steps of a)exposing a sample comprising analytes to a first selectivity conditionto allow retention of analytes by a first adsorbent and to createun-retained sample; b) collecting the un-retained sample comprisinganalytes, exposing the un-retained sample to a second selectivitycondition to allow retention of analytes by a second adsorbent and tocreate un-retained sample; and c) detecting retained analyte under thedifferent selectivity conditions by desorption spectrometry.

[0034] In another aspect this invention provides a substrate fordesorption spectrometry comprising an adsorbent whose bindingcharacteristics vary in a gradient along one or more linear axes.

[0035] In another aspect this invention provides a method forprogressively identifying a selectivity condition with improvedresolution for an analyte in a sample. The method comprises the stepsof: (a) identify a selectivity condition that retains an analyte in asample by (i) exposing a sample to a set of selectivity conditions, eachselectivity condition defined by at least one binding characteristic andat least one elution characteristic; (ii) detecting analyte retainedunder each selectivity condition by desorption spectrometry; and (iii)identifying a selectivity condition that retains the analyte; and (b)identifying a selectivity condition with improved resolution for theanalyte by: (i) selecting at least one binding characteristic or elutioncharacteristic from the identified selectivity condition and adding itto a selectivity characteristic constant set; (ii) exposing the sampleto a modified set of selectivity conditions wherein each selectivitycondition in the modified set comprises (1) the selectivitycharacteristics in the constant set and (2) a binding characteristic orelution characteristic that is not in the constant set; and (iii)identifying a selectivity condition from the modified set by desorptionspectrometry that retains the analyte with improved resolution comparedwith a prior identified selectivity condition. One embodiment comprisesthe step of repeating step (b) at least once. Another embodimentcomprises repeating steps (b) until a selectivity condition isidentified that retains only the target analyte from the sample.

[0036] In another aspect this invention provides a substrate fordesorption spectrometry comprising an adsorbent from a selectivityconditions identified to resolve an analyte by the method of progressiveresolution. In one embodiment the substrate comes in the form of a kitfurther comprising an eluant from the selectivity condition orinstructions on using the eluant in combination with the adsorbent.

[0037] In another aspect this invention provides a method forpreparative purification an analyte from an impure sample. The methodcomprises the steps of a) exposing the sample to a substrate under aplurality of different selectivity conditions; detecting retainedanalyte under the different selectivity conditions by desorptionspectrometry; and identifying selectivity conditions under which theanalyte is retained; b) purifying the analyte by repeating, for aplurality of different identified selectivity conditions, a sequence ofsteps comprising i) exposing the sample to an adsorbent under theidentified selectivity condition to allow retention of the analyte bythe adsorbent; ii) separating the analyte from an impurity that is notretained by the substrate; and iii) collecting the analyte from theadsorbent.

[0038] In another aspect this invention provides a method for preparinga substrate for detecting at least one analyte in a sample. This methodis a combinatorial method for the design and identification ofanalyte-specific adsorbents. It is useful in detecting target analytes.The method comprises the steps of a) exposing the sample to at least twodifferent selectivity conditions, each selectivity condition defined bythe combination of an adsorbent and an eluant, to allow retention of theanalyte by the adsorbent; b) identifying by desorption spectrometry atleast one selectivity condition under which the analyte is retained; andc) preparing a substrate comprising at least one adsorbent of anidentified selectivity condition. In one embodiment, the step ofidentifying comprises identifying at least one selectivity conditionunder which a plurality of analytes are retained. In another embodimentthe step of preparing comprises preparing a substrate comprising aplurality of adsorbents that retain the analyte under an elutioncondition as a multiplex adsorbent.

[0039] In another aspect this invention provides a method of diagnosingin a subject a disease characterized by at least one diagnostic marker.This is a combinatorial method for simultaneous detection of multiplediagnostic markers. The method comprises the steps of a) providing asubstrate for use in desorption spectrometry that comprises at least oneaddressable location, each addressable location comprising an adsorbentthat resolves at least one of the diagnostic markers under an elutioncondition; b) exposing the substrate to a biological sample from thesubject under the elution condition to allow retention of the diagnosticmarker; and c) detecting retained diagnostic marker by desorptionspectrometry. Detecting retained diagnostic marker provides a diagnosisof the disease.

[0040] In another aspect this invention provides a kit for detecting ananalyte in a sample comprising (1) a substrate for use in desorptionspectrometry that comprises at least one addressable location, eachaddressable location comprising an adsorbent that resolves an analyteunder a selectivity condition comprising the adsorbent and an eluant,and (2) the eluant or instructions for exposing the sample to theselectivity condition. In one embodiment the kit is characterized by aplurality of diagnostic markers and the substrate comprises a pluralityof addressable locations, each addressable location comprising anadsorbent that resolves at least one of the diagnostic markers.

[0041] In another aspect this invention provides a substrate fordesorption spectrometry comprising at least one adsorbent in at leastone addressable location wherein the at least one adsorbent resolves aplurality of diagnostic markers for a pathological condition from apatient sample.

[0042] In another aspect this invention provides a method for selectingidentity candidates for an analyte protein. This method is acombinatorial method for protein identification based on at least twophysico-chemical properties. The method comprises the steps of a)determining a value set specifying match parameters for at least a firstand second physico-chemical characteristic of a protein analyte in asample by i) exposing the analyte to a plurality of differentselectivity conditions, wherein adsorption of the protein analyte to thesubstrate is mediated by a basis of attraction that identifies aphysicochemical characteristic of the protein analyte; and ii) detectingretained analyte under the different selectivity conditions bydesorption spectrometry; and b) performing, in a programmable digitalcomputer, the steps of i) accessing a database comprising, for eachmember of a set of reference polypeptides, a value set specifying atleast a first and second physico-chemical characteristic of thereference polypeptides; ii) inputting the value set specifying thephysico-chemical characteristics of the protein analyte; iii) sortingfrom the database, reference polypeptides having value sets within thematch parameters. The sorted reference polypeptides provide identitycandidates for the protein analyte. Unsorted references polypeptides arethose excluded as identity candidates.

[0043] In another aspect this invention provides a method forsequentially retaining analytes. This method is a multimericmacromolecular or supramolecular assembly monitoring method. It isuseful as a method for drug discovery by molecular recognitioninterference. The method comprises the steps of a) exposing a firstsample to a primary adsorbent and to an eluant to allow retention of afirst analyte by the adsorbent, and detecting the adsorbed analyte bydesorption spectrometry, whereby the retained first analyte becomes asecondary adsorbent; b) exposing a second sample to the secondaryadsorbent and to an eluant to allow retention of a second analyte by thesecondary adsorbent, and detecting the adsorbed second analyte bydesorption spectrometry, whereby the retained second analyte becomes atertiary adsorbent.

[0044] In another aspect this invention provides a method of detectingan enzyme in a sample. The method comprises the steps of: a) providing asolid phase comprising an adsorbent and an enzyme substrate bound to theadsorbent, wherein the activity of the enzyme on the enzyme substrateproduces a product having a characteristic molecular mass; b) exposingthe substrate to the sample; and c) detecting the product by desorptionspectrometry. Detecting the product provides a detection of the enzyme.

[0045] In another aspect this invention provides a method fordetermining whether an analyte is differentially present (e.g.,differentially expressed) in a first and second biological sample. Themethod is useful for combinatorial method for differential geneexpression monitoring by differential protein display. The methodcomprises the steps of a) determining a first retention map for theanalyte in the first sample for at least one selectivity condition; b)determining a second retention map for the analyte in the second samplefor the same selectivity condition; and c) detecting a differencebetween the first and the second retention maps. A difference in theretention maps provides a determination that the analyte isdifferentially present in first and second samples.

[0046] In one embodiment the method is for determining whether a proteinis differentially expressed between two different cells, and the firstand second samples comprise the cells or material from the cells. Inanother embodiment the method if for determining whether an agent altersthe expression of a protein in a biological sample further comprisingthe step of administering the agent to a first biological sample but notto a second biological sample. In another embodiment the firstbiological sample derives from a healthy subject and the secondbiological sample is from a subject suffering from a pathologicalcondition. The sample can be selected from, for example, blood, urine,serum and tissue. Analytes that are found to be increased in samplesfrom pathological subjects are candidate diagnostic markers. Generally,confirmation of a dianostic marker involves detection of the marker inmany subjects.

[0047] In another aspect this invention provides a method foridentifying a ligand for a receptor. The method comprises the steps of:a) providing a substrate comprising an adsorbent wherein the receptor isbound to the adsorbent; b) exposing the bound receptor to a samplecontaining the ligand under conditions to allow binding between thereceptor and the ligand; and c) detecting bound ligand by desorptionspectrometry.

[0048] In another aspect this invention provides a screening method fordetermining whether an agent modulates binding between a target analyteand an adsorbent. This is a combinatorial method for drug discovery. Themethod comprises the steps of a) providing a substrate comprising anadsorbent to which the target analyte binds under an elution condition;b) exposing the substrate to the target analyte and to the agent underthe elution condition to allow binding between the target analyte andthe adsorbent; c) detecting an amount of binding between the targetanalyte and the adsorbent by desorption spectrometry; and d) determiningwhether the measured amount is different than a control amount ofbinding when the substrate is exposed to the target analyte under theelution condition without the agent. A difference between the measuredamount and the control amount indicates that the agent modulatesbinding.

[0049] In one aspect, this invention provides a method of detecting agenetic package containing a polynucleotide that encodes a polypeptideagent that specifically binds to a target adsorbent. This is, in oneaspect, a combinatorial method for selecting analyte-specific phage froma display library, including the use of target proteins isolated byretentate mapping or target proteins generated in situ by in vitrotranscription and translation. The method comprises the steps of: a)providing a substrate comprising a target adsorbent; b) providing adisplay library that comprises a plurality of different geneticpackages, each different genetic package comprising a polynucleotidethat comprises a nucleotide sequence that encodes a polypeptide agent,and each different genetic package having a surface on which the encodedpolypeptide agent is displayed; c) exposing the substrate to the displaylibrary under elution conditions to allow specific binding between apolypeptide agent and the target adsorbent, whereby a genetic packagecomprising the polypeptide agent is retained on the substrate; and d)detecting a genetic package retained on the substrate by desorptionspectrometry.

[0050] In one embodiment of this method, the display library is a phagedisplay library. In another embodiment the phage is M13. In anotherembodiment the polypeptide is a single chain antibody. In anotherembodiment the target analyte is a polypeptide analyte that isdifferentially expressed between cells of different phenotypes. Inanother embodiment the substrate comprises a cell or cell membrane.

[0051] In one embodiment, the step of providing the substrate comprisingthe target adsorbent comprises the steps of: i) providing a substratecomprising an adsorbent, wherein the adsorbent retains a target analyteunder an elution condition; and ii) exposing the adsorbent to the targetanalyte under the elution condition to allow retention of the targetanalyte by the adsorbent, whereby the target analyte becomes the targetadsorbent. In one embodiment, the target analyte is a target polypeptideand the step of ii) exposing the adsorbent comprises the step ofproducing the target polypeptide in situ on the adsorbent by in vitrotranslation of a polynucleotide encoding the target polypeptide, and canfurther comprise amplifying the polynucleotide sequence in situ on thesubstrate.

[0052] In another embodiment the substrate comprises (1) an adsorbentthat binds an anchoring polypeptide and (2) at least one target geneticpackage having a surface displaying the anchoring polypeptide and atarget adsorbent polypeptide, the target genetic package comprising apolynucleotide that comprises a nucleotide sequence that encodes thetarget adsorbent, wherein the target genetic package is bound to theadsorbent through the anchoring polypeptide.

[0053] In another embodiment the method further comprises any of thefollowing steps: sequencing the nucleotide sequence that encodes thepolypeptide agent; isolating the retained genetic package or producingthe polypeptide agent.

[0054] In another aspect this invention provides a substrate fordesorption spectrometry comprising an adsorbent that binds an anchoringpolypeptide displayed on a surface of a genetic package, wherein thesurface of the genetic package further displays a target polypeptide andwherein the genetic package comprises a polynucleotide comprising anucleotide sequence that encodes the target polypeptide.

[0055] In another aspect this invention provides a method for detectingtranslation of a polynucleotide. The method comprises the steps of: a)providing a substrate comprising an adsorbent for use in desorptionspectrometry; b) contacting the substrate with the polynucleotideencoding a polypeptide and with agents for in vitro translation of thepolynucleotide, whereby the polypeptide is produced; c) exposing thesubstrate to an eluant to allow retention of the polypeptide by theadsorbent; and d) detecting retained polypeptide by desorptionspectrometry. Detection of the polypeptide provides detection oftranslation of the polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 depicts a substrate containing a plurality of adsorbentspots in the form of a strip. The strip contains six different setsadsorbents classified according to a basis of attraction (hydrophobic,ionic, coordinate covalent and mixed function). The strip containsseveral spots for each type of adsorbent, allowing interrogation of thespots at different times with different eluants, or for archiving andsubsequent analysis.

[0057]FIG. 2 depicts an orthogonal array of adsorbents (surfaceinteraction potentials) in predetermined, addressable locations. Thearray also can take the form of a plate. The array includes variousadsorbents. Upon exposure to the analyte, each strip can be washed by avariety of eluants (selectivity threshold modifiers). Analysis ofretention under different selectivity conditions results in retentionmap or recognition profile.

[0058]FIG. 3 is a representation of the quantitative analysis ofanalytes by desorption of analyte from given locations on the array andquantitative detection of the desorbed analyte by laser desorption massspectrometry.

[0059]FIG. 4A illustrates an example of a computer system used toexecute software that can be used to analyze data generated by thepresent invention. FIG. 4A shows a computer system 1 which includes amonitor 3, screen 5, cabinet 7, keyboard 9, and mouse 11. Mouse 11 mayhave one or more buttons such as mouse buttons 13. Cabinet 7 houses aCD-ROM drive 15 and a hard drive (not shown) that may be utilized tostore and retrieve computer programs including code incorporating thepresent invention. Although a CD-ROM 17 is shown as the computerreadable storage medium, other computer readable storage media includingfloppy disks, DRAM, hard drives, flash memory, tape, and the like may beutilized. Cabinet 7 also houses familiar computer components (not shown)such as a processor, memory, and the like.

[0060]FIG. 4B shows a system block diagram of computer system 1 used toexecute software that can be used to analyze data generated by thepresent invention. As in FIG. 4A, computer system 1 includes monitor 3and keyboard 9. Computer system 1 further includes subsystems such as acentral processor 102, system memory 104, I/O controller 106, displayadapter 108, removable disk 112, fixed disk 116, network interface 118,and speaker 120. Removable disk 112 is representative of removablecomputer readable media like floppies, tape, CD-ROM, removable harddrive, flash memory, and the like. Fixed disk 116 is representative ofan internal hard drive, DRAM, or the like. Other computer systemssuitable for use with the present invention may include additional orfewer subsystems. For example, another computer system could includemore than one processor 102 (i.e., a multi-processor system) or memorycache.

[0061] FIGS. 5A-5F show retention maps for lysozyme under selectivityconditions including six different adsorbents and several differenteluants.

[0062] FIGS. 6A-6B show the resolution at low and high molecular mass ofanalytes in human serum by an immobilized metal adsorbent.

[0063] FIGS. 7A-7B show the resolution at low and high molecular mass ofanalytes in human serum by a variety of adsorbents using the sameeluant.

[0064] FIGS. 8A-8B show the resolution at low and high molecular mass ofanalytes in preterm infant urine by a variety of adsorbents using wateras the eluant.

[0065]FIG. 9 shows resolution of analytes in preterm infant urine usinga hydrophobic phenyl adsorbent and three different eluants, resulting inthe discovery of selective retention of one of the analytes (*) by theTween wash condition.

[0066]FIG. 10A-10D show the resolution of analytes in cell culturemedium of two different breast cancer cell lines.

[0067]FIG. 11 shows a composite retention map of preterm infant urineexposed to selectivity conditions defined by six different adsorbentsand three different eluants.

[0068]FIG. 12 shows a two-dimensional polyacrylamide gel (pI andapparent molecular mass) of preterm infant urine.

[0069]FIG. 13 shows a method of panning with phage display libraries fora phage having a surface protein that specifically binds to a targetanalyte. The substrate depicted at the top shows that even a fewspecifically bound phage can be detected by desorption spectrometrythrough the detection of the many coat proteins that phage contains. Atthe bottom, a substrate with several adsorbent spots is developed sothat the target analyte is specifically bound. Phage are exposed to thespots. Bound phage are detected by desorption spectrometry. Phage boundto another spot can be isolated and grown.

[0070]FIG. 14 shows how a ligand agent, in this case a single chainantibody, identified by a panning method can be used as an adsorbent todock a target protein for use in protein-protein interaction studies. Atarget is purified in situ (spot 2) and used to pan a phage displaylibrary (spot 4). A single chain antibody is isolated and attached to asubstrate (spot 6) as an adsorbent. The target is then adsorbed to thesingle chain antibody. The target is now docked for the study ofprotein-protein interactions (spot 8).

[0071]FIG. 15 shows a method for screening drug candidates for theability to interfere with protein binding to a ligand, in this case asingle-chain antibody. A single chain antibody specific for a targetprotein is docked to a spot on a substrate through, for example, ananti-phage antibody which, itself, can be docked through protein A orprotein G. The single chain antibody is exposed to the target proteinand to drug candidates. The ability of the drug to bind to the analyteprotein and to interfere with ligand binding to analyte is monitored bydesorption spectrometry.

[0072]FIG. 16 shows a method for screening drug candidates for theability to interfere with protein binding to a ligand. The method issimilar to that depicted in the previous figure, except one monitors theability of the drug to interfere with analyte binding by binding,itself, to the ligand by desorption spectrometry.

[0073]FIG. 17 shows a method for screening drug candidates for theability to interfere with target protein (Target protein 1) binding to asecondary ligand (Target Protein II). As in the previous two figures,the target is docked to the substrate becoming, itself, an adsorbent forthe ligand. In this case, the analyte is docked through a single chainantibody. The target is then exposed to the ligand and to the drugcandidates. The ability of the drug to interfere with binding betweenthe analyte and the ligand (by, e.g., binding to the target analyte) ismonitored by desorption spectrometry.

[0074]FIG. 18 depicts a flow chart beginning with the identification ofdifferentially expressed mRNA or polypeptides and ending with thecreation of a diagnostic platform for specifically binding thepolypeptide for detection by desorption spectrometry.

[0075] FIGS. 19A-19D show a retention map of Hemophilus lysate on anadsorbent array. FIG. 19A: anionic adsorbent; FIG. 19B: Normal phaseadsorbent; FIG. 19C: Ni(II) adsorbent; FIG. 19D: Hydrophobic adsorbent.

[0076] FIGS. 20A-20C show progressive resolution of an analyte inHemophilus lysate. The adsorbent in each case was an anionic adsorbent.FIG. 20A: In a first step, after exposure to the sample, the spot waswashed with 150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH7.0. In a second step, the adsorbent and sodium phosphate characteristicof the eluant were added to a constant set of characteristics. A newelution characteristic was added. FIG. 20B: In addition to 20 mM sodiumphosphate, pH 7.0, the spot was washed with 0.05% Triton X100 and 0.15 MNaCl (150 μl, total). FIG. 20C: In addition to 20 mM sodium phosphate,pH 7.0, the spot was washed with 100 mM imidazole, 0.15 M NaCl (150 μltotal).

[0077] FIGS. 21A-21D show the results of a comparison between componentsin normal human serum and diseased serum. FIG. 21A: Retentate map ofnormal serum on an adsorbent array Cu(II) site. FIG. 21B: Retentate mapof disease serum on an adsorbent array Cu(II) site. FIG. 21C: Retainedanalytes of both serum samples are combined in an overlay fashion. Tosimplify the presentation, each peak of retained analyte is converted toa bar, the dashed bars represent analytes retained from a normal serum,and the solid bars represent analytes retained from a disease serum.FIG. 21D: To differentiate more clearly the difference between the twosamples, a comparison plot is generated, where the ratio of the retainedanalytes from the samples are calculated and displayed. The two analytesmarked with “*” show significant increases in the disease serum (5 to 10fold increases).

[0078] FIGS. 22A-22D show a comparison of retentate maps for control,diseased and drug-treated mouse urine on a Cu(II) adsorbent, andquantitation of amount of a marker in diseased and drug-treated urine.

[0079] FIGS. 23A-23D show retentate maps of analytes in urine from fourhuman cancer patients shown in “gel view” format. Difference mapsbetween patients 1, 2 and 3 show two common analytes that are present inincreased amounts in these patients.

[0080] FIGS. 24A-24E show detection of M13 phage by laser desorptionmass spectrometry through the detection of the gene VIII coat protein.The dilutions of the original 10¹² phage per ml range from 1:10 to1:100,000,000.

[0081] FIGS. 25A-25B show the capture of M13 by desorption spectrometryusing anti-M13 antibody as an adsorbent. FIG. 22A shows captured M13phage with peaks representing gene VIII and gene III proteins. FIG. 22Bis a control showing peaks representing the antibody adsorbent (singlyand doubly charged).

[0082] FIGS. 26A-26D show adsorption of M13 phage bearing an anti-tatsingle chain antibody by tat protein adsorbent. Single strength is shownunder phage dilutions from 1:10 to 1:10,000.

[0083] FIGS. 27A-27B show retention maps of TGF-β binding to dockedTGF-β receptor fusion protein at 1 μg/ml (FIG. 27A) and at 100 ng/ml(FIG. 27B). The solid line shows binding without the presence of freeTGF-β receptor. The dashed line shows binding in the presence TGF-βreceptor.

[0084] FIGS. 28 to 31 show the resolving power of retentatechromatography. FIGS. 28A-28C show resolution of proteins fromHemophilus lysate using hydrophobic, cationic and Cu(II) adsorbents atmolecular masses from 0 kD to 30 kD. Each retained analyte isrepresented by a bar, the height of the bar represents the intensity ofthe retained analyte. FIGS. 29A-29C show resolution of proteins fromHemophilus lysate using hydrophobic, cationic and Cu(II) adsorbents atmolecular masses from about 30 kD to about 100 kD. FIG. 30 showscombined resolution from 0 kD to 30 kD of Hemophilus proteins from eachof the three adsorbents. FIG. 31 shows combined resolution from 20 kD to100 kD of Hemophilus proteins from each of the three adsorbents.

[0085]FIG. 32 shows the binding of GST fusion protein to a normaladsorbent.

[0086] FIGS. 33A-33B show binding of a specific ligand to GST fusionreceptor docked to an adsorbent array (FIG. 33A) and lack of binding ofthe ligand to a control array that does not include the GST fusionreceptor (FIG. 33B).

DETAILED DESCRIPTION OF THE INTENTION

[0087] I. Definitions

[0088] Unless defined otherwise, all technical and scientific terms usedherein have the meaning commonly understood by a person skilled in theart to which this invention belongs. The following references provideone of skill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

[0089] “Analyte” refers to a component of a sample which is desirablyretained and detected. The term can refer to a single component or a setof components in the sample.

[0090] “Adsorbent” refers to any material capable of adsorbing ananalyte. The term “adsorbent” is used herein to refer both to a singlematerial (“monoplex adsorbent”) (e.g., a compound or functional group)to which the analyte is exposed, and to a plurality of differentmaterials (“multiplex adsorbent”) to which a sample is exposed. Theadsorbent materials in a multiplex adsorbent are referred to as“adsorbent species.” For example, an addressable location on a substratecan comprise a multiplex adsorbent characterized by many differentadsorbent species (e.g., anion exchange materials, metal chelators, orantibodies), having different binding characteristics.

[0091] “Adsorb” refers to the detectable binding between an absorbentand an analyte either before or after washing with an eluant(selectivity threshold modifier).

[0092] “Substrate” refers to a solid phase to which an adsorbent isattached or deposited.

[0093] “Binding characteristic” refers to a chemical and physicalfeature that dictates the attraction of an adsorbent for an analyte. Twoadsorbents have different binding characteristics if, under the sameelution conditions, the adsorbents bind the same analyte with differentdegrees of affinity. Binding characteristics include, for example,degree of salt-promoted interaction, degree of hydrophobic interaction,degree of hydrophilic interaction, degree of electrostatic interaction,and others described herein.

[0094] “Binding conditions” refer to the binding characteristics towhich an analyte is exposed.

[0095] “Eluant” refers to an agent, typically a solution, that is usedto mediate adsorption of an analyte to an adsorbent. Eluants also arereferred to as “selectivity threshold modifiers.”

[0096] “Elution characteristic” refers to a feature that dictates theability of a particular eluant (selectivity threshold modifier) tomediate adsorption between an analyte and an absorbent. Two eluants havedifferent elution characteristics if, when put in contact with ananalyte and adsorbent, the degree of affinity of the analyte for theadsorbent differs. Elution characteristics include, for example, pH,ionic strength, modification of water structure, detergent strength,modification of hydrophobic interactions, and others described herein.

[0097] “Elution conditions” refer to the elution characteristics towhich an analyte is exposed.

[0098] “Selectivity characteristic” refers to a feature of thecombination of an adsorbent having particular binding characteristicsand an eluant having particular elution characteristics that dictate thespecificity with which the analyte is retained to the adsorbent afterwashing with the eluant.

[0099] “Selectivity conditions” refer to the selectivity characteristicsto which an analyte is exposed.

[0100] “Basis for attraction” refers to the chemical and/orphysico-chemical properties which cause one molecule to be attracted toanother.

[0101] “Strength of attraction” refers to the intensity of theattraction of one molecule for another (also known as affinity).

[0102] “Resolve,” “resolution,” or “resolution of analyte” refers to thedetection of at least one analyte in a sample. Resolution includes thedetection of a plurality of analytes in a sample by separation andsubsequent differential detection. Resolution does not require thecomplete separation of an analyte from all other analytes in a mixture.Rather, any separation that allows the distinction between at least twoanalytes suffices.

[0103] “High information resolution” refers to resolution of an analytein a manner that permits not only detection of the analyte, but also atleast one physico-chemical property of the analyte to be evaluated,e.g., molecular mass.

[0104] “Desorption spectrometry” refers to a method of detecting ananalyte in which the analyte is exposed to energy which desorbs theanalyte from a stationary phase into a gas phase, and the desorbedanalyte or a distinguishable portion of it is directly detected by adetector, without an intermediate step of capturing the analyte on asecond stationary phase.

[0105] “Detect” refers to identifying the presence, absence or amount ofthe object to be detected.

[0106] “Retention” refers to an adsorption of an analyte by an adsorbentafter washing with an eluant.

[0107] “Retention data” refers to data indicating the detection(optionally including detecting mass) of an analyte retained under aparticular selectivity condition.

[0108] “Retention map” refers to a value set specifying retention datafor an analyte retained under a plurality of selectivity conditions.

[0109] “Recognition profile” refers to a value set specifying relativeretention of an analyte under a plurality of selectivity conditions.

[0110] “Complex” refers to analytes formed by the union of 2 or moreanalytes.

[0111] “Fragment” refers to the products of the chemical, enzymatic, orphysical breakdown of an analyte. Fragments may be in a neutral or ionicstate.

[0112] “Differential expression” refers to a detectable difference inthe qualitative or quantitative presence of an analyte.

[0113] “Biological sample” refers to a sample derived from a virus,cell, tissue, organ or organism including, without limitation, cell,tissue or organ lysates or homogenates, or body fluid samples, such asblood, urine or cerebrospinal fluid.

[0114] “Organic biomolecule” refers to an organic molecule of biologicalorigin, e.g., steroids, amino acids, nucleotides, sugars, polypeptides,polynucleotides, complex carbohydrates or lipids.

[0115] “Small organic molecule” refers to organic molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes organic biopolymers (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, up to about 2000 Da, or up to about 1000 Da.

[0116] “Biopolymer” refers to a polymer of biological origin, e.g.,polypeptides, polynucleotides, polysaccharides or polyglycerides (e.g.,di- or tri-glycerides).

[0117] “Polypeptide” refers to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

[0118] “Polynucleotide” refers to a polymer composed of nucleotideunits. Polynucleotides include naturally occurring nucleic acids, suchas deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

[0119] “Detectable moiety” or a “label” refers to a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful labels include ³²P, ³⁵S,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteinsfor which antisera or monoclonal antibodies are available, or nucleicacid molecules with a sequence complementary to a target. The detectablemoiety often generates a measurable signal, such as a radioactive,chromogenic, or fluorescent signal, that can be used to quantitate theamount of bound detectable moiety in a sample. The detectable moiety canbe incorporated in or attached to a primer or probe either covalently,or through ionic, van der Waals or hydrogen bonds, e.g., incorporationof radioactive nucleotides, or biotinylated nucleotides that arerecognized by streptavadin. The detectable moiety may be directly orindirectly detectable. Indirect detection can involve the binding of asecond directly or indirectly detectable moiety to the detectablemoiety. For example, the detectable moiety can be the ligand of abinding partner, such as biotin, which is a binding partner forstreptavadin, or a nucleotide sequence, which is the binding partner fora complementary sequence, to which it can specifically hybridize. Thebinding partner may itself be directly detectable, for example, anantibody may be itself labeled with a fluorescent molecule. The bindingpartner also may be indirectly detectable, for example, a nucleic acidhaving a complementary nucleotide sequence can be a part of a branchedDNA molecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

[0120] “Plurality” means at least two.

[0121] “Purify” or “purification” means removing at least onecontaminant from the composition to be purified. Purification does notrequire that the purified compound be 100% pure.

[0122] A “ligand” is a compound that specifically binds to a targetmolecule.

[0123] A “receptor” is compound that specifically binds to a ligand.

[0124] “Antibody” refers to a polypeptide ligand substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereof,which specifically binds and recognizes an epitope (e.g., an antigen).The recognized immunoglobulin genes include the kappa and lambda lightchain constant region genes, the alpha, gamma, delta, epsilon and muheavy chain constant region genes, and the myriad immunoglobulinvariable region genes. Antibodies exist, e.g., as intact immunoglobulinsor as a number of well characterized fragments produced by digestionwith various peptidases. This includes, e.g., Fab′ and F(ab)′₂fragments. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies. It alsoincludes polyclonal antibodies, monoclonal antibodies, chimericantibodies and humanized antibodies. “Fc” portion of an antibody refersto that portion of an immunoglobulin heavy chain that comprises one ormore heavy chain constant region domains, CH₁, CH₂ and CH₃, but does notinclude the heavy chain variable region.

[0125] A ligand or a receptor (e.g., an antibody) “specifically bindsto” or “is specifically immunoreactive with” a compound analyte when theligand or receptor functions in a binding reaction which isdeterminative of the presence of the analyte in a sample ofheterogeneous compounds. Thus, under designated assay (e.g.,immunoassay) conditions, the ligand or receptor binds preferentially toa particular analyte and does not bind in a significant amount to othercompounds present in the sample. For example, a polynucleotidespecifically binds under hybridization conditions to an analytepolynucleotide comprising a complementary sequence; an antibodyspecifically binds under immunoassay conditions to an antigen analytebearing an epitope against which the antibody was raised; and anadsorbent specifically binds to an analyte under proper elutionconditions.

[0126] “Agent” refers to a chemical compound, a mixture of chemicalcompounds, a sample of undetermined composition, a combinatorial smallmolecule array, a biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal cells or tissues.Suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward et al. (1989) Nature 341: 544-546. The protocoldescribed by Huse is rendered more efficient in combination with phagedisplay technology. See, e.g., Dower et al., WO 91/17271 and McCaffertyet al., WO 92/01047.

[0127] “Recombinant polynucleotide” refers to a polynucleotide havingsequences that are not naturally joined together. An amplified orassembled recombinant polynucleotide may be included in a suitablevector, and the vector can be used to transform a suitable host cell. Ahost cell that comprises the recombinant polynucleotide is referred toas a “recombinant host cell.” The gene is then expressed in therecombinant host cell to produce, e.g., a “recombinant polypeptide.” Arecombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.Appropriate unicellular hosts include any of those routinely used inexpressing eukaryotic or mammalian polynucleotides, including, forexample, prokaryotes, such as E. coli; and eukaryotes, including forexample, fungi, such as yeast; and mammalian cells, including insectcells (e.g., Sf9) and animal cells such as CHO, R1.1, B-W, L-M, AfricanGreen Monkey Kidney cells (e.g. COS 1, COS 7, BSC 1, BSC 40 and BMT 10)and cultured human cells.

[0128] “Expression control sequence” refers to a nucleotide sequence ina polynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked to it.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible, repressible or constitutive), enhancers, transcriptionterminators, a start codon (i.e., ATG), splicing signals for introns,and stop codons.

[0129] “Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

[0130] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA,to serve as templates for synthesis of other polymers and macromoleculesin biological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

[0131] “Energy absorbing molecule” refers to refers to a molecule thatabsorbs energy from an energy source in a desorption spectrometerthereby enabling desorption of analyte from a probe surface. Energyabsorbing molecules used in MALDI are frequently referred to as“matrix.” Cinnamic acid derivatives, cinapinic acid and dihydroxybenzoicacid are frequently used as energy absorbing molecules in laserdesorption of bioorganic molecules.

[0132] II. Retentate Chromatography

[0133] Retentate chromatography is a method for the multidimensionalresolution of analytes in a sample. The method involves (1) selectivelyadsorbing analytes from a sample to a substrate under a plurality ofdifferent adsorbent/eluant combinations (“selectivity conditions”) and(2) detecting the retention of adsorbed analytes by desorptionspectrometry. Each selectivity condition provides a first dimension ofseparation, separating adsorbed analytes from those that are notadsorbed. Desorption mass spectrometry provides a second dimension ofseparation, separating adsorbed analytes from each other according tomass. Because retentate chromatography involves using a plurality ofdifferent selectivity conditions, many dimensions of separation areachieved. The relative adsorption of one or more analytes under the twoselectivity conditions also can be determined. This multidimensionalseparation provides both resolution of the analytes and theircharacterization.

[0134] Further, the analytes thus separated remain docked in a retentatemap that is amenable to further manipulation to examine, for example,analyte structure and/or function. Also, the docked analytes can,themselves, be used as adsorbents to dock other analytes exposed to thesubstrate. In sum, the present invention provides a rapid,multidimensional and high information resolution of analytes.

[0135] The method can take several forms. In one embodiment, the analyteis adsorbed to two different adsorbents at two physically differentlocations and each adsorbent is washed with the same eluant (selectivitythreshold modifier). In another embodiment, the analyte is adsorbed tothe same adsorbent at two physically different locations and washed withtwo different eluants. In another embodiment, the analyte is adsorbed totwo different adsorbents in physically different locations and washedwith two different eluants. In another embodiment, the analyte isadsorbed to an adsorbent and washed with a first eluant, and retentionis detected; then, the adsorbed analyte is washed with a second,different eluant, and subsequent retention is detected.

[0136] A. Methods of Performing Retentate Chromatography

[0137] 1. Exposing The Analyte to Selectivity Conditions

[0138] a. Substrate Preparation

[0139] In performing retentate chromatography an analyte that isretained by an adsorbent is presented to an energy source on asubstrate. A sample containing the analyte may be contacted to theadsorbent before or after the adsorbent is affixed to the substrate thatwill serve to present the analyte to the desorption means. Forcontacting purposes, the adsorbent may be in liquid form or solid form(i.e., on a substrate or solid phase). Specifically, the adsorbent maybe in the form of a solution, suspension, dispersion, water-in-oilemulsion, oil-in-water emulsion, or microemulsion. When the adsorbent isprovided in the form of a suspension, dispersion, emulsion ormicroemulsion, a suitable surfactant may also be present. In thisembodiment, the sample may be contacted with the adsorbent by admixing aliquid sample with the liquid adsorbent. Alternatively, the sample maybe provided on a solid support and contacting will be accomplished bybathing, soaking, or dipping the sample-containing solid support in theliquid adsorbent. In addition, the sample may be contacted by sprayingor washing over the solid support with the liquid adsorbent. In thisembodiment, different adsorbents may be provided in differentcontainers.

[0140] In one embodiment, the adsorbent is provided on a substrate. Thesubstrate can be any material which is capable of binding or holding theadsorbent. Typically, the substrate is comprised of glass; ceramic;electrically conducting polymers (e.g. carbonized PEEK); TEFLON® coatedmaterials; organic polymers; native biopolymers; metals (e.g., nickel,brass, steel or aluminum); films; porous and non-porous beads ofcross-linked polymers (e.g., agarose, cellulose or dextran); otherinsoluble polymers; or combinations thereof.

[0141] In one embodiment, the substrate takes the form of a probe or asample presenting means that is inserted into a desorption detector. Forexample, referring to FIG. 1, the substrate can take the form of astrip. The adsorbent can be attached to the substrate in the form of alinear array of spots, each of which can be exposed to the analyte.Several strips can be joined together so that the plurality ofadsorbents form an array 30 having discrete spots in defined rows. Thesubstrate also can be in the form of a plate having an array ofhorizontal and vertical rows of adsorbents which form a regulargeometric pattern such as a square, rectangle or circle.

[0142] Probes can be produced as follows. The substrate can be any solidmaterial, for example, stainless steel, aluminum or a silicon wafer. Ametal substrate can then be coated with a material that allowsderivitization of the surface. For example a metal surface can be coatedwith silicon oxide, titanium oxide or gold.

[0143] The surface is then derivatized with a bifunctional linker. Thelinker includes at one end a functional group that can covalently bindwith a functional group on the surface. Thus the functional group can bean inorganic oxide or a sulfhydryl group for gold. The other end of thelinker generally has an amino functionality. Useful bifunctional linkersinclude aminopropyl triethoxysilane or aminoethyl disulfide.

[0144] Once bound to the surface, the linkers are further derivatizedwith groups that function as the adsorbent. Generally the adsorbent isadded to addressable locations on the probe. In one type of probe spotsof about 3 mm in diameter are arrange in an orthogonal array. Theadsorbents can, themselves, be part of bifunctional molecules containinga group reactive with the available amino group and the functional groupthat acts as the adsorbent. Functional groups include, for example,normal phase (silicon oxide), reverse phase (C₁₈ aliphatic hydrocarbon),quaternary amine and sulphonate. Also, the surface can be furtherderivatized with other bifunctional molecules such as carbodiimide andN-hydroxysuccinimide, creating a pre-activated blank. These blanks canbe functionalized with bioorganic adsorbents (e.g., nucleic acids,antibodies and other protein ligands). Biopolymers can bind thefunctional groups on the blanks through amine residues or sulfhydrylresidues. In one embodiment, the adsorbents are bound to cross-linkedpolymers (e.g., films) that are themselves bound to the surface of theprobe through the available functional groups. Such polymers include,for example, cellulose, dextran, carboxymethyl dextran, polyacrylamideand mixtures of these. Probes with attached adsorbents are ready foruse.

[0145] In another embodiment, the adsorbent is attached to a firstsubstrate to provide a solid phase, such as a polymeric or glass bead,which is subsequently positioned on a second substrate which functionsas the means for presenting the sample to the desorbing energy of thedesorption detector. For example, the second substrate can be in theform a plate having a series of wells at predetermined addressablelocations. The wells can function as containers for a first substratederivatized with the adsorbent, e.g., polymeric beads derivatized withthe adsorbent. One advantage of this embodiment is that the analyte canbe adsorbed to the first substrate in one physical context, andtransferred to the sample presenting substrate for analysis bydesorption spectrometry.

[0146] Typically, the substrate is adapted for use with the detectorsemployed in the methods of the present invention for detecting theanalyte bound to and retained by the adsorbent. In one embodiment, thesubstrate is removably insertable into a desorption detector where anenergy source can strike the spot and desorb the analyte. The substratecan be suitable for mounting in a horizontally and/or verticallytranslatable carriage that horizontally and/or vertically moves thesubstrate to successively position each predetermined addressablelocation of adsorbent in a path for interrogation by the energy sourceand detection of the analyte bound thereto. The substrate can be in theform of a conventional mass spectrometry probe.

[0147] The strips, plates, or probes of substrate can be produced usingconventional techniques. Thereafter, the adsorbent can be directly orindirectly coupled, fitted, or deposited on the substrate prior tocontacting with the sample containing the analyte. The adsorbent may bedirectly or indirectly coupled to the substrate by any suitable means ofattachment or immobilization. For example, the adsorbent can be directlycoupled to the substrate by derivatizing the substrate with theadsorbent to directly bind the adsorbent to the substrate throughcovalent or non-covalent bonding.

[0148] Attachment of the adsorbent to the substrate can be accomplishedthrough a variety of mechanisms. The substrate can be derivatized with afully prepared adsorbent molecule by attaching the previously preparedadsorbent molecule to the substrate. Alternatively, the adsorbent can beformed on the substrate by attaching a precursor molecule to thesubstrate and subsequently adding additional precursor molecules to thegrowing chain bound to the substrate by the first precursor molecule.This mechanism of building the adsorbent on the substrate isparticularly useful when the adsorbent is a polymer, particularly abiopolymer such as a DNA or RNA molecule. A biopolymer adsorbent can beprovided by successively adding bases to a first base attached to thesubstrate using methods known in the art of oligonucleotide chiptechnology. See, e.g., U.S. Pat. No. 5,445,934 (Fodor et al.).

[0149] As can be seen from FIG. 2, as few as two and as many as 10, 100,1000, 10,000 or more adsorbents can be coupled to a single substrate.The size of the adsorbent site may be varied, depending on experimentaldesign and purpose. However, it need not be larger than the diameter ofthe impinging energy source (e.g., laser spot diameter). The spots cancontinue the same or different adsorbents. In some cases, it isadvantageous to provide the same adsorbent at multiple locations on thesubstrate to permit evaluation against a plurality of different eluantsor so that the bound analyte can be preserved for future use orreference, perhaps in secondary processing. By providing a substratewith a plurality of different adsorbents, it is possible to utilize theplurality of binding characteristics provided by the combination ofdifferent adsorbents with respect to a single sample and thereby bindand detect a wider variety of different analytes. The use of a pluralityof different adsorbents on a substrate for evaluation of a single sampleis essentially equivalent to concurrently conducting multiplechromatographic experiments, each with a different chromatographycolumn, but the present method has the advantage of requiring only asingle system.

[0150] When the substrate includes a plurality of adsorbents, it isparticularly useful to provide the adsorbents in predeterminedaddressable locations. By providing the adsorbents in predeterminedaddressable locations, it is possible to wash an adsorbent at a firstpredetermined addressable location with a first eluant and to wash anadsorbent at a second predetermined addressable location with a secondeluant. In this manner, the binding characteristics of a singleadsorbent for the analyte can be evaluated in the presence of multipleeluants which each selectively modify the binding characteristics of theadsorbent in a different way. The addressable locations can be arrangedin any pattern, but preferably in regular patters, such as lines,orthogonal arrays, or regular curves, such as circles. Similarly, whenthe substrate includes a plurality of different adsorbents, it ispossible to evaluate a single eluant with respect to each differentadsorbent in order to evaluate the binding characteristics of a givenadsorbent in the presence of the eluant. It is also possible to evaluatethe binding characteristics of different adsorbents in the presence ofdifferent eluants.

[0151] (1) Incremental or Gradient Adsorbent Surfaces

[0152] A series of adsorbents having different binding characteristicscan be provided by synthesizing a plurality of different polymericadsorbents on the substrate. The different polymeric adsorbents can beprovided by attaching a precursor molecule to the substrate,initializing the polymerization reaction, and terminating thepolymerization reaction at varied degrees of completion for eachadsorbent. Also, the terminal functional groups in the polymers can bereacted so as to chemically derivatize them to varying degrees withdifferent affinity reagent (e.g., —NH₃, or COO⁻). By terminating thepolymerization or derivatization reaction, adsorbents of varying degreesof polymerization or derivatization are produced. The varying degrees ofpolymerization or derivatization provide different bindingcharacteristics for each different polymeric adsorbent. This embodimentis particularly useful for providing a plurality of different biopolymeradsorbents on a substrate.

[0153] If desired, the polymerization reactions can be carried out in areaction vessel, rather than on the substrate itself. For example,polymeric adsorbents of varying binding characteristics can be providedby extracting an aliquot of product from the reaction vessel as thepolymerization/derivatization reaction is proceeding. The aliquots,having been extracted at various points during thepolymerization/derivatization reaction will exhibit varied degrees ofpolymerization/derivatization to yield a plurality of differentadsorbents. The different aliqouts of product can then be utilized asadsorbents having different binding characteristics. Alternatively, aplurality of different adsorbents can be provided by sequentiallyrepeating the steps of terminating the reaction, withdrawing an aliquotof product, and re-starting the polymerization/derivatization reaction.The products extracted at each termination point will exhibit varyingdegrees of polymerization/derivatization and as a result will provide aplurality of adsorbents having different binding characteristics.

[0154] In one embodiment, a substrate is provided in the form of a stripor a plate that is coated with adsorbent in which one or more bindingcharacteristic varies in a one- or two-dimensional gradient. Forexample, a strip is provided having an adsorbent that is weaklyhydrophobic at one end and strongly hydrophobic at the other end. Or, aplate is provided that is weakly hydrophobic and anionic in one corner,and strongly hydrophobic and anionic in the diagonally opposite corner.Such adsorption gradients are useful in the qualitative analysis of ananalyte. Adsorption gradients can be made by a controlled sprayapplication or by flowing material across a surface in a time-wisemanner to allow incremental completion of a reaction over the dimensionof the gradient. This process can be repeated, at right angles, toprovide orthogonal gradients of similar or different adsorbents withdifferent binding characteristics.

[0155] The sample containing the analyte may be contacted to theadsorbent either before or after the adsorbent is positioned on thesubstrate using any suitable method which will enable binding betweenthe analyte and the adsorbent. The adsorbent can simply be admixed orcombined with the sample. The sample can be contacted to the adsorbentby bathing or soaking the substrate in the sample, or dipping thesubstrate in the sample, or spraying the sample onto the substrate, bywashing the sample over the substrate, or by generating the sample oranalyte in contact with the adsorbent. In addition, the sample can becontacted to the adsorbent by solubilizing the sample in or admixing thesample with an eluant and contacting the solution of eluant and sampleto the adsorbent using any of the foregoing techniques (i.e., bathing,soaking, dipping, spraying, or washing over).

[0156] b. Contacting the Analyte to the Adsorbent

[0157] Exposing the sample to an eluant prior to binding the analyte tothe adsorbent has the effect of modifying the selectivity of theadsorbent while simultaneously contacting the sample to the adsorbent.Those components of the sample which will bind to the adsorbent andthereby be retained will include only those components which will bindthe adsorbent in the presence of the particular eluant which has beencombined with the sample, rather than all components which will bind tothe adsorbent in the absence of elution characteristics which modify theselectivity of the adsorbent.

[0158] The sample should be contacted to the adsorbent for a period oftime sufficient to allow the analyte to bind to the adsorbent.Typically, the sample is contacted with the analyte for a period ofbetween about 30 seconds and about 12 hours. Preferably, the sample iscontacted to the analyte for a period of between about 30 seconds andabout 15 minutes.

[0159] The temperature at which the sample is contacted to the adsorbentis a function of the particular sample and adsorbents selected.Typically, the sample is contacted to the adsorbent under ambienttemperature and pressure conditions, however, for some samples, modifiedtemperature (typically 4° C. through 37° C.) and pressure conditions canbe desirable and will be readily determinable by those skilled in theart.

[0160] Another advantage of the present invention over conventionaldetection techniques is that the present invention enables the numerousdifferent experiments to be conducted on a very small amount of sample.Generally, a volume of sample containing from a few atommoles to 100picomoles of analyte in about 1 μl to 500 μl is sufficient for bindingto the adsorbent. Analyte may be preserved for future experiments afterbinding to the adsorbent because any adsorbent locations which are notsubjected to the steps of desorbing and detecting all of the retainedanalyte will retain the analyte thereon. Therefore, in the case whereonly a very small fraction of sample is available for analysis, thepresent invention provides the advantage of enabling a multitude ofexperiments with different adsorbents and/or eluants to be carried outat different times without wasting sample.

[0161] c. Washing the Adsorbent with Eluants

[0162] After the sample is contacted with the analyte, resulting in thebinding of the analyte to the adsorbent, the adsorbent is washed witheluant. Typically, to provide a multi-dimensional analysis, eachadsorbent location is washed with at least a first and a seconddifferent eluants. Washing with the eluants modifies the analytepopulation retained on a specified adsorbent. The combination of thebinding characteristics of the adsorbent and the elution characteristicsof the eluant provide the selectivity conditions which control theanalytes retained by the adsorbent after washing. Thus, the washing stepselectively removes sample components from the adsorbent.

[0163] The washing step can be carried out using a variety oftechniques. For example, as seen above, the sample can be solubilized inor admixed with the first eluant prior to contacting the sample to theadsorbent. Exposing the sample to the first eluant prior to orsimultaneously with contacting the sample to the adsorbent has, to afirst approximation, the same net effect as binding the analyte to theadsorbent and subsequently washing the adsorbent with the first eluant.After the combined solution is contacted to the adsorbent, the adsorbentcan be washed with the second or subsequent eluants.

[0164] Washing an adsorbent having the analyte bound thereto can beaccomplished by bathing, soaking, or dipping the substrate having theadsorbent and analyte bound thereon in an eluant; or by rinsing,spraying, or washing over the substrate with the eluant. Theintroduction of eluant to small diameter spots of affinity reagent isbest achieved by a microfluidics process.

[0165] When the analyte is bound to adsorbent at only one location and aplurality of different eluants are employed in the washing step,information regarding the selectivity of the adsorbent in the presenceof each eluant individually may be obtained. The analyte bound toadsorbent at one location may be determined after each washing witheluant by following a repeated pattern of washing with a first eluant,desorbing and detecting retained analyte, followed by washing with asecond eluant, and desorbing and detecting retained analyte. The stepsof washing followed by desorbing and detecting can be sequentiallyrepeated for a plurality of different eluants using the same adsorbent.In this manner the adsorbent with retained analyte at a single locationmay be reexamined with a plurality of different eluants to provide acollection of information regarding the analytes retained after eachindividual washing.

[0166] The foregoing method is also useful when adsorbents are providedat a plurality of predetermined addressable locations, whether theadsorbents are all the same or different. However, when the analyte isbound to either the same or different adsorbents at a plurality oflocations, the washing step may alternatively be carried out using amore systematic and efficient approach involving parallel processing.Namely, the step of washing can be carried out by washing an adsorbentat a first location with eluant, then washing a second adsorbent witheluant, then desorbing and detecting the analyte retained by the firstadsorbent and thereafter desorbing and detecting analyte retained by thesecond adsorbent. In other words, all of the adsorbents are washed witheluant and thereafter analyte retained by each is desorbed and detectedfor each location of adsorbent. If desired, after detection at eachadsorbent location, a second stage of washings for each adsorbentlocation may be conducted followed by a second stage of desorption anddetection. The steps of washing all adsorbent locations, followed bydesorption and detection at each adsorbent location can be repeated fora plurality of different eluants. In this manner, and entire array maybe utilized to efficiently determine the character of analytes in asample. The method is useful whether all adsorbent locations are washedwith the same eluant in the first washing stage or whether the pluralityof adsorbents are washed with a plurality of different eluants in thefirst washing stage.

[0167] 2. Detection

[0168] Analytes retained by the adsorbent after washing are adsorbed tothe substrate. Analytes retained on the substrate are detected bydesorption spectrometry: desorbing the analyte from the adsorbent anddirectly detecting the desorbed analytes.

[0169] a. Methods for Desorption

[0170] Desorbing the analyte from the adsorbent involves exposing theanalyte to an appropriate energy source. Usually this means striking theanalyte with radiant energy or energetic particles. For example, theenergy can be light energy in the form of laser energy (e.g., UV laser)or energy from a flash lamp. Alternatively, the energy can be a streamof fast atoms. Heat may also be used to induce/aid desorption.

[0171] Methods of desorbing and/or ionizing analytes for direct analysisare well known in the art. One such method is called matrix-assistedlaser desorption/ionization, or MALDI. In MALDI, the analyte solution ismixed with a matrix solution and the mixture is allowed to crystallizeafter being deposited on an inert probe surface, trapping the analytewithin the crystals may enable desorption. The matrix is selected toabsorb the laser energy and apparently impart it to the analyte,resulting in desorption and ionization. Generally, the matrix absorbs inthe UV range. MALDI for large proteins is described in, e.g., U.S. Pat.No. 5,118,937 (Hillenkamp et al.) and U.S. Pat. No. 5,045,694 (Beavisand Chait).

[0172] Surface-enhanced laser desorption/ionization, or SELDI,represents a significant advance over MALDI in terms of specificity,selectivity and sensitivity. SELDI is described in U.S. Pat. No.5,719,060 (Hutchens and Yip). SELDI is a solid phase method fordesorption in which the analyte is presented to the energy stream on asurface that enhances analyte capture and/or desorption. In contrast,MALDI is a liquid phase method in which the analyte is mixed with aliquid material that crystallizes around the analyte.

[0173] One version of SELDI, called SEAC (Surface-Enhanced AffinityCapture), involves presenting the analyte to the desorbing energy inassociation with an affinity capture device (i.e., an adsorbent). It wasfound that when an analyte is so adsorbed, it can be presented to thedesorbing energy source with a greater opportunity to achieve desorptionof the target analyte. An energy absorbing material can be added to theprobe to aid desorption. Then the probe is presented to the energysource for desorbing the analyte.

[0174] Another version of SELDI, called SEND (Surface-Enhanced NeatDesorption), involves the use of a layer of energy absorbing materialonto which the analyte is placed. A substrate surface comprises a layerof energy absorbing molecules chemically bond to the surface and/oressentially free of crystals. Analyte is then applied alone (i.e., neat)to the surface of the layer, without being substantially mixed with it.The energy absorbing molecules, as do matrix, absorb the desorbingenergy and cause the analyte to be desorbed. This improvement issubstantial because analytes can now be presented to the energy sourcein a simpler and more homogeneous manner because the performance ofsolution mixtures and random crystallization is eliminated. Thisprovides more uniform and predictable results that enable automation ofthe process. The energy absorbing material can be classical matrixmaterial or can be matrix material whose pH has been neutralized orbrought into the basic range. The energy absorbing molecules can bebound to the probe through covalent or noncovalent means.

[0175] Another version of SELDI, called SEPAR (Surface-EnhancedPhotolabile Attachment and Release), involves the use of photolabileattachment molecules. A photolabile attachment molecule is a divalentmolecule having one site covalently bound to a solid phase, such a flatprobe surface or another solid phase, such as a bead, that can be madepart of the probe, and a second site that can be covalently bound withthe affinity reagent or analyte. The photolabile attachment molecule,when bound to both the surface and the analyte, also contains aphotolabile bond that can release the affinity reagent or analyte uponexposure to light. The photolabile bond can be within the attachmentmolecule or at the site of attachment to either the analyte (or affinityreagent) or the probe surface.

[0176] b. Method for Direct Detection of Analytes

[0177] The desorbed analyte can be detected by any of several means.When the analyte is ionized in the process of desorption, such as inlaser desorption/ionization mass spectrometry, the detector can be anion detector. Mass spectrometers generally include means for determiningthe time-of-flight of desorbed ions. This information is converted tomass. However, one need not determine the mass of desorbed ions toresolve and detect them: the fact that ionized analytes strike thedetector at different times provides detection and resolution of them.

[0178] Alternatively, the analyte can be detectably labeled with, e.g.,a fluorescent moiety or with a radioactive moiety. In these cases, thedetector can be a fluorescence or radioactivity detector.

[0179] A plurality of detection means can be implemented in series tofully interrogate the analyte components and function associated withretentate at each location in the array.

[0180] c. Desorption Detectors

[0181] Desorption detectors comprise means for desorbing the analytefrom the adsorbent and means for directly detecting the desorbedanalyte. That is, the desorption detector detects desorbed analytewithout an intermediate step of capturing the analyte in another solidphase and subjecting it to subsequent analysis. Detection of an analytenormally will involve detection of signal strength. This, in turn,reflects the quantity of analyte adsorbed to the adsorbent.

[0182] Beyond these two elements, the desorption detector also can haveother elements. One such element is means to accelerate the desorbedanalyte toward the detector. Another element is means for determiningthe time-of-flight of analyte from desorption to detection by thedetector.

[0183] A preferred desorption detector is a laser desorption/ionizationmass spectrometer, which is well known in the art. The mass spectrometerincludes a port into which the substrate that carries the adsorbedanalytes, e.g., a probe, is inserted. Desorption is accomplished bystriking the analyte with energy, such as laser energy. The device caninclude means for translating the surface so that any spot on the arrayis brought into line with the laser beam. Striking the analyte with thelaser results in desorption of the intact analyte into the flight tubeand its ionization. The flight tube generally defines a vacuum space.Electrified plates in a portion of the vacuum tube create an electricalpotential which accelerate the ionized analyte toward the detector. Aclock measures the time of flight and the system electronics determinesvelocity of the analyte and converts this to mass. As any person skilledin the art understands, any of these elements can be combined with otherelements described herein in the assembly of desorption detectors thatemploy various means of desorption, acceleration, detection, measurementof time, etc.

[0184] B. Selectivity Conditions

[0185] One advantage of the invention is the ability to expose theanalytes to a variety of different binding and elution conditions,thereby providing both increased resolution of analytes and informationabout them in the form of a recognition profile. As in conventionalchromatographic methods, the ability of the adsorbent to retain theanalyte is directly related to the attraction or affinity of the analytefor the adsorbent as compared to the attraction or affinity of theanalyte for the eluant or the eluant for the adsorbent. Some componentsof the sample may have no affinity for the adsorbent and therefore willnot bind to the adsorbent when the sample is contacted to the adsorbent.Due to their inability to bind to the adsorbent, these components willbe immediately separated from the analyte to be resolved. However,depending upon the nature of the sample and the particular adsorbentutilized, a number of different components can initially bind to theadsorbent.

[0186] 1. Adsorbents

[0187] Adsorbents are the materials that bind analytes. A plurality ofadsorbents can be employed in retentate chromatography. Differentadsorbents can exhibit grossly different binding characteristics,somewhat different binding characteristics, or subtly different bindingcharacteristics. Adsorbents which exhibit grossly different bindingcharacteristics typically differ in their bases of attraction or mode ofinteraction. The basis of attraction is generally a function of chemicalor biological molecular recognition. Bases for attraction between anadsorbent and an analyte include, for example, (1) a salt-promotedinteraction, e.g., hydrophobic interactions, thiophilic interactions,and immobilized dye interactions; (2) hydrogen bonding and/or van derWaals forces interactions and charge transfer interactions, such as inthe case of a hydrophilic interactions; (3) electrostatic interactions,such as an ionic charge interaction, particularly positive or negativeionic charge interactions; (4) the ability of the analyte to formcoordinate covalent bonds (i.e., coordination complex formation) with ametal ion on the adsorbent; (5) enzyme-active site binding; (6)reversible covalent interactions, for example, disulfide exchangeinteractions; (7) glycoprotein interactions; (8) biospecificinteractions; or (9) combinations of two or more of the foregoing modesof interaction. That is, the adsorbent can exhibit two or more bases ofattraction, and thus be known as a “mixed functionality” adsorbent.

[0188] a. Salt-promoted Interaction Adsorbents

[0189] Adsorbents which are useful for observing salt-promotedinteractions include hydrophobic interaction adsorbents. Examples ofhydrophobic interaction adsorbents include matrices having aliphatichydrocarbons, specifically C₁-C₁₈ aliphatic hydrocarbons; and matriceshaving aromatic hydrocarbon functional groups such as phenyl groups.Hydrophobic interaction adsorbents bind analytes which include unchargedsolvent exposed amino acid residues, and specifically amino acidresidues which are commonly referred to as nonpolar, aromatic andhydrophobic amino acid residues, such as phenylalanine and tryptophan.Specific examples of analytes which will bind to a hydrophobicinteraction adsorbent include lysozyme and DNA. Without wishing to bebound by a particular theory, it is believed that DNA binds tohydrophobic interaction adsorbents by the aromatic nucleotides in DNA,specifically, the purine and pyrimidine groups.

[0190] Another adsorbent useful for observing salt-promoted interactionsincludes thiophilic interaction adsorbents, such as for example T-GEL®which is one type of thiophilic adsorbent commercially available fromPierce, Rockford, Ill. Thiophilic interaction adsorbents bind, forexample, immunoglobulins such as IgG. The mechanism of interactionbetween IgG and T-GEL® is not completely known, but solvent exposed trpresidues are suspected to play a role.

[0191] A third adsorbent which involves salt-promoted ionic interactionsand also hydrophobic interactions includes immobilized dye interactionadsorbents. Immobilized dye interaction adsorbents include matrices ofimmobilized dyes such as for example CIBACHRON™ blue available fromPharmacia Biotech, Piscataway, N.J. Immobilized dye interactionadsorbents bind proteins and DNA generally. One specific example of aprotein which binds to an immobilized dye interaction adsorbent isbovine serum albumin (BSA).

[0192] b. Hydrophilic Interaction Adsorbents

[0193] Adsorbents which are useful for observing hydrogen bonding and/orvan der Waals forces on the basis of hydrophilic interactions includesurfaces comprising normal phase adsorbents such as silicon-oxide (i.e.,glass). The normal phase or silicon-oxide surface, acts as a functionalgroup. In addition, adsorbents comprising surfaces modified withhydrophilic polymers such as polyethylene glycol, dextran, agarose, orcellulose can also function as hydrophilic interaction adsorbents. Mostproteins will bind hydrophilic interaction adsorbents because of a groupor combination of amino acid residues (i.e., hydrophilic amino acidresidues) that bind through hydrophilic interactions involving hydrogenbonding or van der Waals forces. Examples of proteins which will bindhydrophilic interaction adsorbents include myoglobin, insulin andcytochrome C.

[0194] In general, proteins with a high proportion of polar or chargedamino acids will be retained on a hydrophilic surface. Alternatively,glycoproteins with surface exposed hydrophilic sugar moieties, also havehigh affinity for hydrophilic adsorbents.

[0195] c. Electrostatic Interaction Adsorbents

[0196] Adsorbents which are useful for observing electrostatic or ioniccharge interactions include anionic adsorbents such as, for example,matrices of sulfate anions (i.e., SO₃ ⁻) and matrices of carboxylateanions (i.e., COO⁻) or phosphate anions (OPO₃ ⁻). Matrices havingsulfate anions are permanent negatively charged. However, matriceshaving carboxylate anions have a negative charge only at a pH abovetheir pKa. At a pH below the pKa, the matrices exhibit a substantiallyneutral charge. Suitable anionic adsorbents also include anionicadsorbents which are matrices having a combination of sulfate andcarboxylate anions and phosphate anions. The combination provides anintensity of negative charge that can be continuously varied as afunction of pH. These adsorbents attract and bind proteins andmacromolecules having positive charges, such as for example ribonucleaseand lactoferrin. Without wishing to be bound by a particular theory, itis believed that the electrostatic interaction between an adsorbent andpositively charged amino acid residues including lysine residues,arginine residues, and histidyl residues are responsible for the bindinginteraction.

[0197] Other adsorbents which are useful for observing electrostatic orionic charge interactions include cationic adsorbents. Specific examplesof cationic adsorbents include matrices of secondary, tertiary orquaternary amines. Quaternary amines are permanently positively charged.However, secondary and tertiary amines have charges that are pHdependent. At a pH below the pKa, secondary and tertiary amines arepositively charged, and at a pH above their pKa, they are negativelycharged. Suitable cationic adsorbents also include cationic adsorbentswhich are matrices having combinations of different secondary, tertiary,and quaternary amines. The combination provides an intensity of positivecharge that can be continuously varied as a function of pH. Cationicinteraction adsorbents bind anionic sites on molecules includingproteins having solvent exposed amino acid residues, such as asparticacid and glutamic acid residues.

[0198] In the case of ionic interaction adsorbents (both anionic andcationic) it is often desirable to use a mixed mode ionic adsorbentcontaining both anions and cations. Such adsorbents provide a continuousbuffering capacity as a function of pH. The continuous bufferingcapacity enables the exposure of a combination of analytes to eluantshaving differing buffering components especially in the pH range of from2 to 11. This results in the generation of local pH environments on theadsorbent which are defined by immobilized titratable proton exchangegroups. Such systems are equivalent to the solid phase separationtechnique known as chromatofocusing. Follicle stimulating hormoneisoforms, which differ mainly in the charged carbohydrate components areseparated on a chromatofocusing adsorbent.

[0199] Still other adsorbents which are useful for observingelectrostatic interactions include dipole-dipole interaction adsorbentsin which the interactions are electrostatic but no formal charge ortitratable protein donor or acceptor is involved.

[0200] d. Coordinate Covalent Interaction Adsorbents

[0201] Adsorbents which are useful for observing the ability to formcoordinate covalent bonds with metal ions include matrices bearing, forexample, divalent and trivalent metal ions. Matrices of immobilizedmetal ion chelators provide immobilized synthetic organic molecules thathave one or more electron donor groups which form the basis ofcoordinate covalent interactions with transition metal ions. The primaryelectron donor groups functioning as immobilized metal ion chelatorsinclude oxygen, nitrogen, and sulfur. The metal ions are bound to theimmobilized metal ion chelators resulting in a metal ion complex havingsome number of remaining sites for interaction with electron donorgroups on the analyte. Suitable metal ions include in general transitionmetal ions such as copper, nickel, cobalt, zinc, iron, and other metalions such as aluminum and calcium. Without wishing to be bound by anyparticular theory, metals ions are believed to interact selectively withspecific amino acid residues in peptides, proteins, or nucleic acids.Typically, the amino acid residues involved in such interactions includehistidine residues, tyrosine residues, tryptophan residues, cysteineresidues, and amino acid residues having oxygen groups such as asparticacid and glutamic acid. For example, immobilized ferric ions interactwith phosphoserine, phosphotyrosine, and phosphothreonine residues onproteins. Depending on the immobilized metal ion, only those proteinswith sufficient local densities of the foregoing amino acid residueswill be retained by the adsorbent. Some interactions between metal ionsand proteins can be so strong that the protein cannot be severed fromthe complex by conventional means. Human β casein, which is highlyphosphorylated, binds very strongly to immobilized Fe(III). Recombinantproteins which are expressed with a 6-Histidine tag, binds very stronglyto immobilized Cu(II) and Ni(II).

[0202] e. Enzyme-Active Site Interaction Adsorbents

[0203] Adsorbents which are useful for observing enzyme-active sitebinding interactions include proteases (such as trypsin), phosphatases,kinases, and nucleases. The interaction is a sequence-specificinteraction of the enzyme binding site on the analyte (typically abiopolymer) with the catalytic binding site on the enzyme. Enzymebinding sites of this type include, for example, active sites of trypsininteracting with proteins and peptides having lysine-lysine orlysine-arginine pairs in their sequence. More specifically, soybeantrypsin inhibitor interacts with and binds to an adsorbent ofimmobilized trypsin. Alternatively, serine proteases are selectivelyretained on immobilized L-arginine adsorbent.

[0204] f. Reversible Covalent Interaction Adsorbents

[0205] Adsorbents which are useful for observing reversible covalentinteractions include disulfide exchange interaction adsorbents.Disulfide exchange interaction adsorbents include adsorbents comprisingimmobilized sulfhydryl groups, e.g., mercaptoethanol or immobilizeddithiothrietol. The interaction is based upon the formation of covalentdisulfide bonds between the adsorbent and solvent exposed cysteineresidues on the analyte. Such adsorbents bind proteins or peptideshaving cysteine residues and nucleic acids including bases modified tocontain reduced sulfur compounds.

[0206] g. Glycoprotein Interaction Adsorbents

[0207] Adsorbents which are useful for observing glycoproteininteractions include glycoprotein interaction adsorbents such asadsorbents having immobilize lectins (i.e., proteins bearingoligosaccharides) therein, an example of which is CONCONAVALIN™, whichis commercially available from Pharmacia Biotech of Piscataway, N.J.Such adsorbents function on the basis of the interaction involvingmolecular recognition of carbohydrate moieties on macromolecules.Examples of analytes which interact with and bind to glycoproteininteraction adsorbents include glycoproteins, particularlyhistidine-rich glycoproteins, whole cells and isolated subcellularfractions.

[0208] h. Biospecific Interaction Adsorbents

[0209] Adsorbents which are useful for observing biospecificinteractions are generically termed “biospecific affinity adsorbents.”Adsorption is considered biospecific if it is selective and the affinity(equilibrium dissociation constant, Kd) is at least 10⁻³ M to (e.g.,10⁻⁵ M, 10⁻⁷ M, 10⁻⁹ M). Examples of biospecific affinity adsorbentsinclude any adsorbent which specifically interacts with and binds aparticular biomolecule. Biospecific affinity adsorbents include forexample, immobilized antibodies which bind to antigens; immobilized DNAwhich binds to DNA binding proteins, DNA, and RNA; immobilizedsubstrates or inhibitors which bind to proteins and enzymes; immobilizeddrugs which bind to drug binding proteins; immobilized ligands whichbind to receptors; immobilized receptors which bind to ligands;immobilized RNA which binds to DNA and RNA binding proteins; immobilizedavidin or streptavidin which bind biotin and biotinylated molecules;immobilized phospholipid membranes and vesicles which bind lipid-bindingproteins. Enzymes are useful adsorbents that can modify an analyteadsorbent thereto. Cells are useful as adsorbents. Their surfacespresent complex binding characteristics. Adsorption to cells is usefulfor identifying, e.g., ligands or signal molecules that bind to surfacereceptors. Viruses or phage also are useful as adsorbents. Virusesfrequently have ligands for cell surface receptors (e.g., gp120 forCD4). Also, in the form a phage display library, phage coat proteins actas agents for testing binding to targets. Biospecific interactionadsorbents rely on known specific interactions such as those describedabove. Other examples of biospecific interactions for which adsorbentscan be utilized will be readily apparent to those skilled in the art andare contemplated by the present invention.

[0210] In one embodiment, the biospecific adsorbent can further comprisean auxiliary, or “helper”, molecule that does not directly participatein binding the target analyte.

[0211] i. Degrees of Binding Specificity

[0212] By exposure to adsorbents having different modes of interaction,the components of a sample can be grossly divided based upon theirinteraction with the different adsorbents. Thus, the attraction of theanalyte for adsorbents having different modes of interaction provides afirst separation parameter. For example, by exposing a sample containingthe analyte to a first adsorbent with a basis of attraction involvinghydrophobicity and a second adsorbent with a basis of attractioninvolving ionic charge, it is possible to separate from the sample thoseanalytes which bind to a hydrophobic adsorbent and to separate thoseanalytes which bind to an adsorbent having the particular ionic charge.

[0213] Adsorbents having different bases of attraction provideresolution of the analyte with a low degree of specificity because theadsorbent will bind not only the analyte, but any other component in thesample which also exhibits an attraction for the adsorbent by the samebasis of attraction. For example, a hydrophobic adsorbent will bind notonly a hydrophobic analyte, but also any other hydrophobic components inthe sample; a negatively charged adsorbent will bind not only apositively charged analyte, but also any other positively chargedcomponent in the sample; and so on.

[0214] The resolution of analytes based upon the basis of attraction ofthe analyte for the adsorbent can be further refined by exploitingbinding characteristics of relatively intermediate specificity oraltered strength of attraction. Resolution of the analyte on the basisof binding characteristics of intermediate specificity can beaccomplished, for example, by utilizing mixed functionality adsorbents.Once the resolution of the analyte is accomplished with relatively lowspecificity, the binding characteristic found to attract the analyte ofinterest can be exploited in combination with a variety of other bindingand elution characteristics to remove still more undesired componentsand thereby resolve the analyte.

[0215] For example, if the analyte binds to hydrophobic adsorbents, theanalyte can be further resolved from other hydrophobic sample componentsby providing a mixed functionality adsorbent which exhibits as one basisof attraction a hydrophobic interaction and also exhibits a second,different basis of attraction. The mixed functionality adsorbent mayexhibit hydrophobic interactions and negatively charged ionicinteractions so as to bind hydrophobic analytes which are positivelycharged. Alternatively, the mixed functionality adsorbent can exhibithydrophobic interactions and the ability to form coordinate covalentbonds with metal ions so as to bind hydrophobic analytes having theability to form coordination complexes with metal ions on the adsorbent.Still further examples of adsorbents exhibiting binding characteristicsof intermediate specificity will be readily apparent to those skilled inthe art based upon the disclosure and examples set forth above.

[0216] The resolution of analytes on the basis of bindingcharacteristics of intermediate specificity can be further refined byexploiting binding characteristics of relatively high specificity.Binding characteristics of relatively high specificity can be exploitedby utilizing a variety of adsorbents exhibiting the same basis ofattraction but a different strength of attraction. In other words,although the basis of attraction is the same, further resolution of theanalyte from other sample components can be achieved by utilizingadsorbents having different degrees of affinity for the analyte.

[0217] For example, an analyte that binds an adsorbent based upon theanalyte's acidic nature may be further resolved from other acidic samplecomponents by utilizing adsorbents having affinity for analytes inspecific acidic pH ranges. Thus the analyte may be resolved using oneadsorbent attracted to sample components of pH 1-2, another adsorbentattracted to sample components of pH of 3-4, and a third adsorbentattracted to sample components of pH of 5-6. In this manner, an analytehaving a specific affinity for an adsorbent which binds analyte of pH of5-6 will be resolved from sample components of pH of 1-4. Adsorbents ofincreasing specificity can be utilized by decreasing the interval ofattraction, i.e., the difference between the binding characteristics ofadsorbents exhibiting the same basis of attraction.

[0218] A primary analyte adsorbed to a primary adsorbent can, itself,have adsorbent properties. In this case, the primary analyte adsorbed toa substrate can become a secondary adsorbent for isolating secondaryanalytes. In turn, the retained secondary analyte can function as atertiary adsorbent to isolate a tertiary analyte from a sample. Thisprocess can continue through several iterations.

[0219] 2. Eluants

[0220] The eluants, or wash solutions, selectively modify the thresholdof absorption between the analyte and the adsorbent. The ability of aneluant to desorb and elute a bound analyte is a function of its elutioncharacteristics. Different eluants can exhibit grossly different elutioncharacteristics, somewhat different elution characteristics, or subtlydifferent elution characteristics.

[0221] The temperature at which the eluant is contacted to the adsorbentis a function of the particular sample and adsorbents selected.Typically, the eluant is contacted to the adsorbent at a temperature ofbetween 0° C. and 100° C., preferably between 4° C. and 37° C. However,for some eluants, modified temperatures can be desirable and will bereadily determinable by those skilled in the art.

[0222] As in the case of adsorbents, eluants which exhibit grosslydifferent elution characteristics generally differ in their basis ofattraction. For example, various bases of attraction between the eluantand the analyte include charge or pH, ionic strength, water structure,concentrations of specific competitive binding reagents, surfacetension, dielectric constant and combinations of two or more of theabove.

[0223] a. pH-Based Eluants

[0224] Eluants which modify the selectivity of the adsorbent based uponpH (i.e., charge) include known pH buffers, acidic solutions, and basicsolutions. By washing an analyte bound to a given adsorbent with aparticular pH buffer, the charge can be modified and therefore thestrength of the bond between the adsorbent and the analyte in thepresence of the particular pH buffer can be challenged. Those analyteswhich are less competitive than others for the adsorbent at the pH ofthe eluant will be desorbed from the adsorbent and eluted, leaving boundonly those analytes which bind more strongly to the adsorbent at the pHof the eluant.

[0225] b. Ionic Strength-Based Eluants

[0226] Eluants which modify the selectivity of the adsorbent withrespect to ionic strength include salt solutions of various types andconcentrations. The amount of salt solubilized in the eluant solutionaffects the ionic strength of the eluant and modifies the adsorbentbinding ability correspondingly. Eluants containing a low concentrationof salt provide a slight modification of the adsorbent binding abilitywith respect to ionic strength. Eluants containing a high concentrationof salt provide a greater modification of the adsorbent binding abilitywith respect to ionic strength.

[0227] c. Water Structure-Based Eluants

[0228] Eluants which modify the selectivity of the adsorbent byalteration of water structure or concentration include urea andchaotropic salt solutions. Typically, urea solutions include, e.g.,solutions ranging in concentration from 0.1 to 8 M. Chaotropic saltswhich can be used to provide eluants include sodium thiocyanate. Waterstructure-based eluants modify the ability of the adsorbent to bind theanalyte due to alterations in hydration or bound water structure.Eluants of this type include for example, glycerol, ethylene glycol andorganic solvents. Chaotropic anions increase the water solubility ofnonpolar moieties thereby decreasing hydrophobic interactions betweenthe analyte and the adsorbent.

[0229] d. Detergent-Based Eluants

[0230] Eluants which modify the selectivity of the adsorbent withrespect to surface tension and analyte structure include detergents andsurfactants. Suitable detergents for use as eluants include ionic andnonionic detergents such as CHAPS, TWEEN and NP-40. Detergent-basedeluants modify the ability of the adsorbent to bind the analyte as thehydrophobic interactions are modified when the hydrophobic andhydrophilic groups of the detergent are introduced. Hydrophobicinteractions between the analyte and the adsorbent, and within theanalyte are modified and charge groups are introduced, e.g., proteindenaturation with ionic detergents such as SDS.

[0231] e. Hydrophobicity-Based Eluants

[0232] Eluants which modify the selectivity of the adsorbent withrespect to dielectric constant are those eluants which modify theselectivity of the adsorbent with respect to hydrophobic interaction.Examples of suitable eluants which function in this capacity includeurea (0.1-8M) organic solvents such as propanol, acetonitrile, ethyleneglycol and glycerol, and detergents such as those mentioned above. Useof acetonitrile as eluant is typical in reverse phase chromatography.Inclusion of ethylene glycol in the eluant is effective in elutingimmunoglobulins from salt-promoted interactions with thiophilicadsorbents.

[0233] f. Combinations of Eluants

[0234] Suitable eluants can be selected from any of the foregoingcategories or can be combinations of two or more of the foregoingeluants. Eluants which comprise two or more of the foregoing eluants arecapable of modifying the selectivity of the adsorbent for the analyte onthe basis of multiple elution characteristics.

[0235] 3. Variability of Two Parameters

[0236] The ability to provide different binding characteristics byselecting different adsorbents and the ability to provide differentelution characteristics by washing with different eluants permitsvariance of two distinct parameters each of which is capable ofindividually effecting the selectivity with which analytes are bound tothe adsorbent. The fact that these two parameters can be varied widelyassures a broad range of binding attraction and elution conditions sothat the methods of the present invention can be useful for binding andthus detecting many different types of analytes.

[0237] The selection of adsorbents and eluants for use in analyzing aparticular sample will depend on the nature of the sample, and theparticular analyte or class of analytes to be characterized, even if thenature of the analytes are not known. Typically, it is advantageous toprovide a system exhibiting a wide variety of binding characteristicsand a wide variety of elution characteristics, particularly when thecomposition of the sample to be analyzed is unknown. By providing asystem exhibiting broad ranges of selectivity characteristics, thelikelihood that the analyte of interest will be retained by one or moreof the adsorbents is significantly increased.

[0238] One skilled in the art of chemical or biochemical analysis iscapable of determining the selectivity conditions useful for retaining aparticular analyte by providing a system exhibiting a broad range ofbinding and elution characteristics and observing binding and elutioncharacteristics which provide the best resolution of the analyte.Because the present invention provides for systems including broadranges of selectivity conditions, the determination by one skilled inthe art of the optimum binding and elution characteristics for a givenanalyte can be easily accomplished without the need for undueexperimentation.

[0239] C. Analytes

[0240] The present invention permits the resolution of analytes basedupon a variety of biological, chemical, or physico-chemical propertiesof the analyte by exploiting the properties of the analyte through theuse of appropriate selectivity conditions. Among the many properties ofanalytes which can be exploited through the use of appropriateselectivity conditions are the hydrophobic index (or measure ofhydrophobic residues in the analyte), the isoelectric point (i.e., thepH at which the analyte has no charge), the hydrophobic moment (ormeasure of amphipathicity of an analyte or the extent of asymmetry inthe distribution of polar and nonpolar residues), the lateral dipolemoment (or measure of asymmetry in the distribution of charge in theanalyte), a molecular structure factor (accounting for the variation insurface contour of the analyte molecule such as the distribution ofbulky side chains along the backbone of the molecule), secondarystructure components (e.g., helix, parallel and antiparallel sheets),disulfide bands, solvent-exposed electron donor groups (e.g., His),aromaticity (or measure of pi-pi interaction among aromatic residues inthe analyte) and the linear distance between charged atoms.

[0241] These are representative examples of the types of propertieswhich can be exploited for the resolution of a given analyte from asample by the selection of appropriate selectivity characteristics inthe methods of the present invention. Other suitable properties ofanalytes which can form the basis for resolution of a particular analytefrom the sample will be readily known and/or determinable by thoseskilled in the art and are contemplated by the instant invention.

[0242] The inventive method is not limited with respect to the types ofsamples which can be analyzed. Samples can be in the solid, liquid, orgaseous state, although typically the sample will be in a liquid state.Solid or gaseous samples are preferably solubilized in a suitablesolvent to provide a liquid sample according to techniques well withinthe skill of those in the art. The sample can be a biologicalcomposition, non-biological organic composition, or inorganiccomposition. The technique of the present invention is particularlyuseful for resolving analytes in a biological sample, particularlybiological fluids and extracts; and for resolving analytes innon-biological organic compositions, particularly compositions of smallorganic and inorganic molecules.

[0243] The analytes may be molecules, multimeric molecular complexes,macromolecular assemblies, cells, subcellular organelles, viruses,molecular fragments, ions, or atoms. The analyte can be a singlecomponent of the sample or a class of structurally, chemically,biologically, or functionally related components having one or morecharacteristics (e.g., molecular weight, isoelectric point, ioniccharge, hydrophobic/hydrophilic interaction, etc.) in common.

[0244] Specific examples of analytes which may be resolved using theretentate chromatography methods of the present invention includebiological macromolecules such as peptides, proteins, enzymes,polynucleotides, oligonucleotides, nucleic acids, carbohydrates,oligosaccharides, polysaccharides; fragments of biologicalmacromolecules set forth above, such as nucleic acid fragments, peptidefragments, and protein fragments; complexes of biological macromoleculesset forth above, such as nucleic acid complexes, protein-DNA complexes,receptor-ligand complexes, enzyme-substrate, enzyme inhibitors, peptidecomplexes, protein complexes, carbohydrate complexes, and polysaccharidecomplexes; small biological molecules such as amino acids, nucleotides,nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones,amides, amines, carboxylic acids, vitamins and coenzymes, alcohols,aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growthregulators, phosphate esters and nucleoside diphospho-sugars, syntheticsmall molecules such as pharmaceutically or therapeutically effectiveagents, monomers, peptide analogs, steroid analogs, inhibitors,mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores,antimetabolites, amino acid analogs, antibacterial agents, transportinhibitors, surface-active agents (surfactants), mitochondrial andchloroplast function inhibitors, electron donors, carriers andacceptors, synthetic substrates for proteases, substrates forphosphatases, substrates for esterases and lipases and proteinmodification reagents; and synthetic polymers, oligomers, and copolymerssuch as polyalkylenes, polyamides, poly(meth)acrylates, polysulfones,polystyrenes, polyethers, polyvinyl ethers, polyvinyl esters,polycarbonates, polyvinyl halides, polysiloxanes, POMA, PEG, andcopolymers of any two or more of the above.

[0245] III. Information Processing

[0246] Detection of analytes adsorbed to an adsorbent under particularelution conditions provides information about analytes in a sample andtheir chemical character. Adsorption depends, in part, upon the bindingcharacteristics of the adsorbent: Analytes that bind to an adsorbentpossess the characteristic that makes binding possible. For example,molecules that are cationic at a particular pH will bind to an anionicadsorbent under elution conditions that include that pH. Stronglycationic molecules will only be eluted from the adsorbent under verystrong elution conditions. Molecules with hydrophobic regions will bindto hydrophobic adsorbents, while molecules with hydrophilic regions willbind to hydrophilic adsorbents. Again, the strength of the interactionwill depend, in part, upon extent to which an analyte containshydrophobic or hydrophilic regions. Thus, the determination that certainanalytes in a sample bind to an adsorbent under certain elutionconditions not only resolves analytes in a mixture by separating themfrom each other and from analytes that do not possess the appropriatechemical character for binding, but also identifies a class of analytesor individual analytes having the particular chemical character.Collecting information about analyte retention on one or more particularadsorbents under a variety of elution conditions provides not onlydetailed resolution of analytes in a mixture, but also chemicalinformation about the analytes, themselves that can lead to theiridentity. This data is referred to as “retention data.”

[0247] Data generated in retention assays is most easily analyzed withthe use of a programmable digital computer. The computer programgenerally contains a readable medium that stores codes. Certain code isdevoted to memory that includes the location of each feature on asubstrate array, the identity of the adsorbent at that feature and theelution conditions used to wash the adsorbent. Using this information,the program can then identify the set of features on the array definingcertain selectivity characteristics. The computer also contains codethat receives as input, data on the strength of the signal at variousmolecular masses received from a particular addressable location on theprobe. This data can indicate the number of analytes detected,optionally including for each analyte detected the strength of thesignal and the determined molecular mass.

[0248] The computer also contains code that processes the data. Thisinvention contemplates a variety of methods for processing the data. Inone embodiment, this involves creating an analyte recognition profile.For example, data on the retention of a particular analyte identified bymolecular mass can be sorted according to a particular bindingcharacteristic, for example, binding to anionic adsorbents orhydrophobic adsorbents. This collected data provides a profile of thechemical properties of the particular analyte. Retention characteristicsreflect analyte function which, in turn, reflects structure. Forexample, retention to coordinate covalent metal chelators can reflectthe presence of histidine residues in a polypeptide analyte. Using dataof the level of retention to a plurality of cationic and anionicadsorbents under elution at a variety of pH levels reveals informationfrom which one can derive the isoelectric point of a protein. This, inturn, reflects the probable number of ionic amino acids in the protein.Accordingly, the computer can include code that transforms the bindinginformation into structural information. Furthermore, secondaryprocessing of the analyte (e.g., post-translational modifications)results in an altered recognition profile reflected by differences inbinding or mass.

[0249] In another embodiment, retention assays are performed under thesame set of selectivity thresholds on two different cell types, and theretention data from the two assays is compared. Differences in theretention maps (e.g., presence or strength of signal at any feature)indicate analytes that are differentially expressed by the two cells.This can include, for example, generating a difference map indicatingthe difference in signal strength between two retention assays, therebyindicating which analytes are increasingly or decreasingly retained bythe adsorbent in the two assays.

[0250] The computer program also can include code that receivesinstructions from a programmer as input. The progressive and logicalpathway for selective desorption of analytes from specified,predetermined locations in the array can be anticipated and programmedin advance.

[0251] The computer can transform the data into another format forpresentation. Data analysis can include the steps of determining, e.g.,signal strength as a function of feature position from the datacollected, removing “outliers” (data deviating from a predeterminedstatistical distribution), and calculating the relative binding affinityof the analytes from the remaining data.

[0252] The resulting data can be displayed in a variety of formats. Inone format, the strength of a signal is displayed on a graph as afunction of molecular mass. In another format, referred to as “gelformat,” the strength of a signal is displayed along a linear axisintensity of darkness, resulting in an appearance similar to bands on agel. In another format, signals reaching a certain threshold arepresented as vertical lines or bars on a horizontal axis representingmolecular mass. Accordingly, each bar represents an analyte detected.Data also can be presented in graphs of signal strength for an analytegrouped according to binding characteristic and/or elutioncharacteristic.

[0253] IV. Applications of Retentate Chromatography

[0254] Retentate chromatography involves a combinatorial separationmethod, including detection and characterization of multiple analytes inparallel. These combinatorial methods have many applications. Suchapplications include, without limitation, developing target analytedetection schemes; developing protein purification strategies; proteinpurification methods; identifying specific phage from a phage displaylibrary that bind to a target analyte, including target epitopeidentification using complementary phage display libraries; proteinidentification based on physico-chemical properties of the analyte; geneexpression monitoring and differential protein display; toxicologyscreening; simultaneous detection of multiple diagnostic markers; drugdiscovery; multimeric protein assembly monitoring and detection of invitro polynucleotide translation.

[0255] A. Methods for Sequentially Extracting Analytes from a Sample

[0256] Retentate chromatography involves the analysis of retention of ananalyte under a plurality of adsorbent/eluent conditions. One variationof this method is sequential extraction. In sequential extraction asample is not independently exposed to two different selectivityconditions. Rather, the sample is exposed to a first selectivitycondition to extract certain analytes from the sample onto theadsorbent, and leave non-adsorbed analytes in the eluent. Then, theeluent is exposed to a second selectivity condition. This furtherextracts various analytes from the eluant. Frequently, if the adsorbentsin the first and second exposure have different basis for attraction(e.g., normal phase and hydrophobic) the adsorbent will extract adifferent set of analytes from the eluent. This second eluant is thenexposed to a third selectivity condition, and so on. In one method ofpracticing sequential extraction, the adsorbent is placed at the bottomof a well so that sample can be mixed on top of it. An eluant is addedto the adsorbent and after allowing binding between analytes in thesample, the eluant wash is collected. The collected wash is then exposedto a second adsorbent, and analytes are extracted from the sample bybinding.

[0257] In one embodiment, the goal of sequential extraction ispreparative rather than analytical. More specifically, the goal may beto extract all but a desired analyte from the sample. In this case, thesample is usually small, e.g., a few microliters on a spot about a fewmillimneters in diameter. The adsorbents are selected so as not toadsorb an analyte one wishes not to be depleted from the sample. Afterseveral iterations the finally collected wash is depleted of un-desiredanalytes, leaving the desired ones for subsequent analysis by, forexample, desorption spectrometry or traditional chromatographic methods.

[0258] In another embodiment, unretained sample is, itself, analyzed foranalytes by any analytic technique. Even after a single retention step,this process allows one to examine materials adsorbed to an adsorbentand those analytes that are not adsorbed.

[0259] B. Methods for Progressive Resolution of Analytes in a Sample

[0260] One object of retentate chromatography is the unambiguousresolution of an analyte from a complex sample mixture. This isespecially important for applications in clinical diagnostics, drugdiscovery and functional genomics: These areas can involve theidentification of one or more analytes from a biological sample. Thisinvention provides a method for identifying selectivity conditions withimproved resolution for an analyte. The method involves identifying aselectivity condition in which the analyte is retained and, in aniterative process, adding additional binding characteristics or elutioncharacteristics to the selectivity condition which provide improvedresolution of the analyte.

[0261] A mass spectrum of a complex sample exposed to a selectivitycondition generally includes signals from many components of the sample.The complexity of the signals may interfere with unambiguous resolutionof the analyte. Methods for progressive resolution of an analyte allowone to identify selectivity conditions with improved resolution of theanalyte for unambiguous detection of an analyte in a sample. Aselectivity condition exhibits “improved resolution” of an analytecompared with another selectivity condition if the analyte signal ismore easily distinguishable from the signals of other components. Thiscan include, for example, decreasing the number of analytes bound to theadsorbent, thereby decreasing the total number of signals, or increasingthe selectivity of the selectivity condition for the analyte, therebyenhancing the analyte signal compared with other signals. Of course,when the analyte is exclusively bound to the substrate, it generates thesole analyte signal during detection.

[0262] Methods of progressive resolution involve an iterative process inwhich additional selectivity (binding or elution) characteristics aresequentially added to a constant set of selectivity characteristicsknown to retain the analyte. In a first step a series of selectivityconditions are tested to identify one that retains the target analyte.In a next step, one or more of the selectivity characteristics of theselectivity condition are selected for the constant set for furtheranalysis.

[0263] A new set of selectivity conditions is generated. Each of theconditions in the new set includes the selected characteristics in theconstant set, and at least one new condition not in the constant set.For example, if the constant set includes an anionic adsorbent and a lowsalt eluant, the new condition could involve varying the pH of theeluant. Each of these new variables is tested for the ability to improvethe resolution of the analyte, and one modified selectivity conditionwith improved resolution is identified. In a next step, an addedselectivity condition that provides improved resolution is added to theconstant set.

[0264] The modified constant set is tested again in the same way, bygenerating a new set of selectivity conditions that include thecharacteristics of the constant set and a set of new characteristics.Thus, at each step, the selectivity conditions are selected so thatresolution of the analyte is improved compared with the selectivitycondition at a previous step.

[0265] The method is well described by example. A cell sample typicallycontains hundreds or perhaps thousands of proteins. One may wish toobtain unambiguous resolution of a single target protein analyte in thesample. In a first step, a retention map is developed for the targetanalyte using a plurality of selectivity conditions. For example, theadsorbents could be an anion exchanger, a cation exchanger, a normalphase adsorbent and a reverse phase adsorbent. The elution conditionstested on each adsorbent could be a variety of pH levels, a variety ofionic strengths, a variety of detergent conditions and a variety ofhydrophobicity-based conditions. For example, four different elutionconditions could be tested for each condition. Thus, in this example,sixteen different selectivity conditions are tested for their ability toadsorb the target analyte.

[0266] From this retention map one selects at least one selectivitycondition under which the target analyte is retained. One may select aselectivity condition under which the target bound maximally. However,it may be advantageous to select a condition under which the target isnot maximally bound if this selectivity condition is more selective forthe target than the other selectivity conditions. Presume, for thisexample, that analysis of the retention map shows that the target isretained by anion exchange adsorbents at around neutral pH, but also isweakly adsorbed to a hydrophilic adsorbent.

[0267] One variable absorbent or eluant from the selectivity conditionidentified to result in retention of the analyte is then selected foruse on all subsequent selectivity conditions. As used herein, it is saidto be added to the “set of selectivity condition constants.”

[0268] In the next iteration, one tests the ability of the targetanalyte to bind under a second plurality of selectivity conditions. Eachselectivity condition at the second set includes the elements of theselectivity condition constant set. However, each selectivity alsoincludes another variable—a different adsorbent or eluant added to theselectivity condition. Thus, within the constraint of employing at leastthe set of constants, the second set of selectivity conditions also arechosen to be more diverse than the first set. Methods of increasing thediversity include, for example, testing finer gradations of an elutioncondition or different strengths of an adsorbent. It also can include,for example, the addition of another selectivity characteristic into theselectivity conditions.

[0269] Continuing the example, the anion exchange adsorbent may be addedto the set of constants. This condition is now tested with a widervariety of variables, e.g. eluants or adsorbents. Eluants to be testedcan include a variety of low ph buffers at finer gradations than testedin the first iteration. For example, the first iteration may have testedbuffers at pH 3.0, pH 5.0, pH 7.0 and pH 9.0, and showed that the targetbound to the anion exchange adsorbent near neutral pHs. During thesecond iteration, the buffers tested could be at pH 5.0, pH 5.5, pH 6.0,pH 6.5, pH 7.0, pH 7.5 and pH 8.0. In addition, each of these buffersalso could be varied to include other elution characteristics, e.g.,ionic strength, hydrophobicity, etc.

[0270] Analysis of the second retention map resulting at this stagegenerally will allow one to identify a condition that provided betterresolution than the selectivity condition identified in the first round.Again, one of the variables of this selectivity condition is chosen andadded to the set of selectivity condition constants for furtherinterrogation in the next iteration.

[0271] Continuing the example, suppose the selectivity condition in thesecond round that resolves the analyte best uses a buffer at pH 6.5.This eluant can now be added to the set of constants, which now includesan anion exchange resin and a pH 6.5 buffer. In the next iteration, theselectivity conditions include this constant set, and another variable.The variable might be, for example, addition of a new component to theeluant, such as different ionic strengths; or another adsorbent can beadded into the mixture, such as variety of hydrophobic adsorbents mixedwith the anion exchange adsorbent; or one may vary the density of theanion exchange resin. Again, a selectivity condition is identified fromthis set that shows improved resolution of the analyte.

[0272] The process can continue until the analyte is purified toessential homogeneity. In this case, the selectivity condition isspecific for the analyte.

[0273] As one can see, by increasing the number of variables tested ateach step, one can decrease the number of iterations needed to identifya suitable selectivity condition.

[0274] C. Methods of Preparative Purification of an Analyte

[0275] In another aspect, this invention provides methods of purifyingan analyte. The methods take advantage of the power of retentatechromatography to rapidly identify bases of attraction for adsorbing ananalyte. A first step involves exposing the analyte to a plurality ofselectivity conditions and determining retention under the conditions byretentate chromatography. This generates a recognition profilecharacteristic of the analyte. The selectivity conditions under whichthe analyte is retained are used to develop a protocol for preparativepurification of the analyte.

[0276] For preparative purification of the analyte, the analyte issequentially adsorbed and eluted from a series of adsorbent/eluantcombinations that were identified as binding the analyte. Thus, forexample, the recognition map may indicate that the analyte binds to anormal phase adsorbent and to a metal chelator. The analyte is thencontacted with a first chromatography column, for example, containingthe normal phase adsorbent, which binds the analyte. Unbound material iswashed off. Then the analyte is eluted by a sufficiently stringent wash.The eluant is then contacted with a metal chelate column, for example,to bind the analyte. Unbound materials are washed away. Then, the boundmaterial that includes the analyte, is eluted from the metal chelatecolumn. In this way, the analyte is isolated in preparative amounts. Apreparative amount of a sample is at least 10 μl, at least 100 μl, atleast 1 ml or at least 10 ml.

[0277] The information generated during progressive resolution ofanalytes can be used to design larger scale chromatographic(elution-based) protein purification strategies. The adsorbent bases forattraction, the binding conditions, and the elution conditions (i.e.,the selectivity conditions) for a target analyte protein become definedby retentate chromatography. This information can save an enormousamount of time, energy, and precious analyte that would otherwise bewasted during the trial and error process of purification strategydesign that is now in place. This section also provides for large scalepurification efforts performed with commercially available adsorbents.

[0278] D. Methods for Making Probes for Specific Detection of Analytes

[0279] This invention provides probes for the specific detection of oneor more analytes by desorption spectrometry, as well as methods forgenerating these probes. Such analyte-resolving probes are useful thespecific detection of analytes in diagnostic and analytic methods.

[0280] The first step in generating a probe for resolving one or moreanalytes is to produce a retention map for the analytes under aplurality of different adsorbent/eluant combinations. For example, theresolution of the analytes can be determined for four differentadsorbents washed with each of five different eluants. This providestwenty sets of retention data for each of the analytes. Analysis of theresulting retention map will indicate which selectivity condition orconditions best resolves the analytes. Preferably, one selectivitycondition can be identified that unambiguously resolves all theanalytes. Then, one or more selectivity conditions is selected for usein the analyte-resolving probe so that each of the analytes is resolvedon at least one adsorbent spot. The probe also could contain anadsorbent that does not bind the analyte or analytes. This adsorbentspot is useful as a control. The probes can include a plurality ofadsorbent spots in addressable locations selected for their ability toretain and resolve the analyte or analyte. In this case, adsorbents areselected that bind the analyte under a single eluant condition. This isuseful because the entire probe can be washed with a single eluant inthe detection process.

[0281] The retention map generated for a particular analyte can be usedcreate a customized adsorbent for the analyte. For example, the natureof the adsorbents that retain an analyte indicate a set of bases forattraction of an analyte. A customized adsorbent can be designed bypreparing a multiplex adsorbent that includes elements of adsorbentsthat provide these bases for attraction. Such a custom adsorbent is veryselective for the target analyte. One or a few custom adsorbents cansuffice to generate a recognition map for the analyte. For example, ifit is found that under particular elution conditions an analyte isretained by adsorbents that bind materials that have certain degrees ofhydrophobicity, positive charge and aromaticity, one can create a customadsorbent by design or through the use of combinatorial syntheticstrategies having functional groups that attract each of these threecharacteristics. Detecting binding to this adsorbent identifies theanalyte.

[0282] Such probes are useful for detecting the analyte or analytes in asample. The sample is exposed to the selectivity conditions and theprobe is interrogated by desorption spectrometry. Because the proberesolves the analytes, their presence can be detected by looking for thecharacteristic recognition profile. Such probes are particularly usefulfor identifying a set of diagnostic markers in a patient sample.

[0283] In one embodiment, the array is designed to dock specific classesof protein of interest. This includes diagnostic markers as well asanalytes defined by function. For example, an array can be prepared thatspecifically docks cell surface proteins, enzymes of a certain class(e.g., kinases), transcription factors, intracellular receptors, etc.The adsorbents can be specific for the biopolymers, for example,antibodies.

[0284] In one embodiment, the adsorbents are genetic packages such asphage displaying protein ligands for a certain class of proteins. Inthis case, a phage display library can be pre-screened with a certainclass of molecules to eliminate phage that bind to that class. Then,phage that have been subtracted from the population are used asadsorbents.

[0285] E. Diagnostic Probes and Methods of Diagnosis

[0286] Diagnosis of pathological conditions frequently involves thedetection in a patient sample of one or more molecular markers ofdisease. Certain conditions can be diagnosed by the presence of a singlediagnostic marker. Diagnosis of other conditions may involve detectionof a plurality diagnostic markers. Furthermore, the detection of severalmarkers may increase the confidence of diagnosis. Accordingly, thisinvention provides probes for desorption spectrometry comprising atleast one adsorbent that resolves at least one diagnostic marker of apathological condition.

[0287] The preparation of such probes involves, first, the selection ofmarkers to be detected. The marker can be a marker for any diseasestate, e.g., cancer, heart disease, autoimmune disease, viral infection,Alzheimer's disease or diabetes. For example, detection of prostatespecific antigen (PSA) is highly suggestive of prostate cancer. HIVinfection can be diagnosed by detecting antibodies against several HIVproteins, such as one of p17, p24 or p55 and one of p31, p51 or p66 andone of gp41 or gp120/160. Detection of amyloid-β42 and tau protein incerebrospinal fluid is highly indicative of Alzheimer's disease. Also,the markers can be identified by methods of this invention involvingdetecting differential presence of an analyte in healthy subjects versussubjects with pathological conditions.

[0288] In a next step, adsorbents are developed that retain one or morediagnostic markers. Preferably, a single adsorbent is prepared thatresolves all the markers. This can be accomplished, for example, bycreating a spot containing several antibodies, each of which binds oneof the desired markers. Alternatively, the probe can comprise aplurality of adsorbent spots, each spot capable of resolving at leastone target analyte under a selectivity condition. In one embodiment, theadsorbent is a multiplex adsorbent comprising ligands that are specificfor the markers. For example, the adsorbent can comprise an antibody, apolypeptide ligand or a polynucleotide that specifically binds thetarget analyte. In one embodiment, the antibody is a single chainantibody identified by screening a combinatorial library. Single chainantibodies that are specific for particular markers can be developed byscreening phage display libraries by methods described herein.

[0289] In another embodiment, the adsorbent comprises non-organicbiomolecular components that either retain the target analytespecifically or that retain the analyte with sufficient specificity forunambiguous resolution by desorption spectrometry. Preparation ofadsorbents for detection of specific analytes also are described herein.

[0290] Significantly, a single adsorbent spot used in these methods neednot be specific for a single analyte and, therefore, need not requirebiopolymer-mediated specific affinity between target and adsorbent.Prior affinity detection methods have relied mainly on specific bindingbetween a biopolymer and a target. This includes, for example, thespecific affinity of an antibody for a protein, a polynucleotide for acomplementary polynucleotide or a lectin for a carbohydrate. Suchspecificity was necessary because these means of detection wereindirect: the target was not identified; a label, frequently bound tothe adsorbent, was identified. Accordingly, the more specific theadsorbent, the less likelihood that contaminants would bind to theadsorbent and interfere with specific detection. However, desorptionspectrometry results in direct detection of an analyte. Accordingly, thepresence of contaminants does not interfere with specific detectionunless the signal of the contaminant overlaps with the signal of thetarget.

[0291] Methods of diagnosis involve, first, selecting a patient sampleto be tested. The sample can be, e.g., tissue, blood, urine, stool orother bodily fluid (lymph, cerebrospinal, interarticular, etc.). Then,the sample is exposed to a substrate containing the diagnosticadsorbents under conditions to allow retention of the diagnosticmarkers. The adsorbent is washed with an appropriate eluant. Then themarkers are detected (e.g., resolved) by desorption spectrometry (e.g.,mass spectrometry).

[0292] This invention also provides kits for specific detection ofdiagnostic markers including (1) a substrate for use in desorptionspectrometry that comprises at least one adsorbent in at least oneaddressable location that resolves at least one diagnostic marker undera selectivity condition that comprises the adsorbent and an eluant and(2) the eluant or instructions for preparation of the eluant. Uponexposing the sample to the adsorbent and washing with the eluant, i.e.,by executing the selectivity condition, the analyte is sufficientlypurified or specifically bound for resolution by desorptionspectrometry.

[0293] F. Methods for Identifying Proteins

[0294] In another aspect, this invention provides a method for aiding inthe identification of a protein. The method involves determining matchparameters for physico-chemical characteristics of a protein analyteusing retentate chromatography and searching a protein database toidentify proteins having the match parameters. The derivation ofphysico-chemical information based on retention characteristics isdiscussed above. The database typically will provide the amino acidsequence and/or the nucleotide sequence encoding the amino acid sequenceof each protein. Structural characteristics, such as molecular mass,hydrophobicity, pI, fragment mass, etc. are easily derivable from thisinformation. An analyte protein will share any particular structuralcharacteristic with only a subset of the proteins in the database.Accordingly, identity candidates are found by sorting the proteinsaccording to structural characteristics shared with the protein analyte.Thus, in view of the inaccuracy, degree of specificity, or level ofconfidence inherent in identifying one or more physicochemicalproperties of the reference, one cannot expect that proteins in thedatabase will perfectly match all the characteristics of the reference.Accordingly, the match parameters can be set to identify, for example,the closeness of fit between the protein analyte characteristics and thecharacteristics of the reference polypeptides in the database.

[0295] As our identification of genes in the genome increases, thechance that any protein analyte exists in the database as a referencepolypeptide also increases. Accordingly, this method enables one torapidly resolve a protein of interest in a sample, obtain structuralinformation about the protein, and then use this information to identifythe protein.

[0296] G. Methods for Assembling Multimeric Molecules

[0297] The ability of adsorbents to dock desired molecules is useful inbuilding multimeric molecules and assessing compounds that effect theirassembly. A unit of the multimeric molecule is bound to an adsorbent.Then it is exposed to a sample that contains another unit of themultimeric molecule. Expose can be performed under a variety ofconditions to test binding parameters. The binding of a subunit to themultimer can be monitored by desorption spectrometry. Then, a subsequentsubunit can be tested for binding in the same way. The drug screeningmethods described herein are useful for testing agents for the abilityto interfere with assembly. Accordingly, an analyte at one stage of theprocess becomes an adsorbent at the next stage.

[0298] H. Methods for Performing Enzyme Assays

[0299] This invention also provides methods for performing enzymeassays. Enzyme assays generally involve exposing a sample to be testedwith an enzyme substrate under conditions under which the enzyme isactive. After allowing the enzyme to act on the substrate, a product ofthe enzymatic reaction is detected. In quantitative assays, the amountof product is determined. This amount usually is compared to a controlor a standard curve, thereby yielding an amount of enzyme activity inthe sample.

[0300] This invention provides methods for detecting an enzyme,including detecting an amount of enzyme activity, in a sample. Themethod takes advantage of the fact that the activity of an enzyme oftenproduces a product whose mass is different than the original substrate.In the method, a solid phase is prepared that comprises an adsorbentthat binds the substrate. An amount of the substrate is bound to theadsorbent. Then the adsorbent is exposed to the sample under conditionsand for a time that allows any enzyme to act on the substrate. Then, anybound material is detected by desorption spectrometry. Detection of ananalyte having a molecular mass characteristic of the product of enzymeactivity provides an indication of the presence of the enzyme. Thesignal strength will be a function of the amount of enzymatic activityin the sample.

[0301] I. Methods for Identifying Analytes that are DifferentiallyExpressed Between Biological Materials

[0302] In another aspect this invention provides methods for identifyingorganic biomolecules, particularly proteins, that are differentiallyexpressed between two or more samples. “Differential expression” refersto differences in the quantity or quality of an analyte between twosamples. Such differences could result at any stage of proteinexpression from transcription through post-translational modification.The methods take advantage of the extraordinary resolving power andsensitivity of retentate chromatography. First, recognition profilesusing the same set of selectivity conditions are prepared for analytesfrom the two biological samples. The greater the number of selectivityconditions used, the greater the resolution of analytes in the sampleand, therefore, the greater the number of analytes that can be compared.Then, the recognition maps are compared to identify analytes that aredifferentially retained by the two sets of adsorbents. Differentialretention includes quantitative retention. This indicates, e.g., up- ordown-regulation of expression. Differential retention also includesqualitative differences in the analyte. For example, differences inpost-translational modification of a protein can result in differencesin recognition maps detectable as differences in binding characteristics(for example, if the protein is glycosylated, it will bind differentlyto lectin adsorbents) or differences in mass (for example, as a resultof differences in post-translational cleavage) The analysis can becarried out by a programmable, digital computer.

[0303] The method is particularly useful to detect genes that aredifferentially expressed between two cell types. The two cell typescould be normal versus pathologic cells, e.g., cancer cells or cells atdifferent levels or cells at different stages of development ordifferentiation, or in different parts of the cell cycle. However, themethod also is useful in examining two cells of the same type exposed todifferent conditions. For example, the method is useful in toxicologyscreening and testing agents for the ability to modulate gene expressionin a cell. In such a method, one biological sample is exposed to thetest agent, and other cell is not. Then, retentate maps of the samplesare compared. This method may indicate that a protein or otherbiomolecule is increased or decreased in expression, or is changed someway based on different retention characteristics or different mass.

[0304] Using information about the physico-chemical properties ofdifferentially expressed proteins obtained from the retention maps,identity candidates for these proteins can be determined using methodsdescribed herein.

[0305] This method is useful for identifying diagnostic markers ofdisease. Proteins that are differentially expressed in a patient sampleor a diseased cultured cell compared to normal samples or cells may bediagnostic markers. In general, it is best to compare samples from astatistically significant patient population with normal samples. Inthis way, information can be pooled to identify diagnostic markerscommon to all or a significant number of individuals exhibiting thepathology.

[0306] 1. Increasing Sensitivity by Catabolic Signal Amplification

[0307] The sensitivity of detecting differential presence (e.g,resulting from differential expression) of large proteins in a complexmixture can be increased significantly by fragmenting the large proteininto smaller pieces and detecting the smaller pieces. Increasedsensitivity is due to several factors. First, when all the proteins in asample are fragmented by, for example, enzymatic digestion, largeproteins are likely to produce more fragments than small proteins.Second, the overall sensitivity of desorption spectrometry is greater atlower molecular masses than higher molecular masses. Third, fragmentinga protein increases the number of signals from that target, therebyincreasing the likelihood of detecting that target. Fourth, fragmentinga protein increases the likelihood of capturing and, therefore,detecting, at least one fragment of the protein. Fifth, if a protein isdifferentially present in two samples, then by increasing the number ofsignals from that protein, the difference in amount is more likely to bedetected.

[0308] Also, the method is counter-intuitive. Generally, one seeks todecrease the complexity of an analyte mixture before analysis.Fragmentation increases the complexity.

[0309] Accordingly, in one embodiment of this invention the sensitivityof detecting an analyte is increased by converting the analyte intolower molecular mass fragments before detection. Fragmentation can beachieved by any means known in the art. For example, protein analytescan be fragmented using endoproteases. Carbohydrate analytes can befragmented using glycosidases. Nucleic acids can be fragmented usingendonucleases. The sample can be subject to fragmentation before orafter docking with the adsorbent.

[0310] J. Methods for Identifying Ligands for a Receptor

[0311] Functional pathways in biological systems frequently involve theinteraction between a receptor and a ligand. For example, the bindingbetween transcriptional activation frequently involves the prior bindingof a ligand with a transcription factor. Many pathological conditionsinvolve abnormal interaction between a receptor and its ligand.Interruption of the binding between a receptor and a ligand is afrequent target of drug discovery. However, the identity of a ligand fora receptor frequently is unknown; the receptor is an “orphan” receptor.

[0312] This invention provides a method using retentate chromatographyto identify ligands for receptors. The method involves docking areceptor to an adsorbent. Then, a sample that is suspected of containinga ligand for the receptor is exposed to the docked receptor under anelution condition appropriate for binding between the receptor and theligand. Then, ligands that have bound to the receptor are detected bydesorption spectrometry. The power of this method derives, in part, fromthe sensitivity to desorption spectrometry to detect small quantities ofmaterial docked to an adsorbent.

[0313] Docking the receptor to the adsorbent requires identifying anadsorbent that retains, and preferably, specifically binds, thereceptor. Methods for identifying adsorbents that specifically bind aprotein are described herein. In one method, the adsorbent comprises anantibody specific for the receptor. In another embodiment, the receptoris produced as recombinant fusion protein that includes a moiety forspecific binding. For example, the receptor can be fused with the Fcportion of an antibody. Such portions bind to protein A which can beincorporated into an adsorbent.

[0314] The sample tested for the presence of a ligand is at thediscretion of the practitioner. For example, if the receptor is anuclear receptor, the sample can be nuclear extract. If the receptor isa cytoplasmic receptor, the sample can be cytoplasmic extract. If thereceptor is a cell surface receptor, the sample can be fluid from thesurface to which the cell is exposed, for example, serum for anepithelial cell surface receptor.

[0315] The sample generally will be incubated with the receptor underphysiological conditions for a time sufficient to allow binding, forexample 37° C. for several hours. Then, unbound material is washed away.This method can quickly identify ligands that conventional techniquesrequire months to identify.

[0316] Retentate chromatography allows parallel processing of samples onseveral adsorbent spots. Accordingly, this method can involve testing aplurality of different samples for the presence of a ligand, as well asthe testing of a single sample under a plurality of incubation andelution conditions.

[0317] By determining the mass of the identified ligand and variousphysico-chemical properties, the ligand can be positively identifiedusing information from genome databases.

[0318] In another embodiment of this method a set of probes is preparedwhich has been exposed to and has docked proteins from a cell. Thisprobe is useful, itself, as a secondary probe to identify molecules fromthe cell that bind to the docked molecules. After preparing a retentatemap from the probe, the probe is secondarily exposed to the testmaterial, generally under less stringent conditions than those used toprepare the secondary probe, and the addressable locations analysed.Molecules that are newly docked to the probe are those bound to thealready-docked molecules.

[0319] K. Methods for Drug Discovery

[0320] Identifying molecules that intervene in the binding between areceptor and its ligand is an important step in developing drugs. Thisinvention provides methods of screening compounds for their ability tomodulate the binding between an adsorbent and an analyte (e.g., areceptor adsorbent and a ligand analyte) by exposing an adsorbent andanalyte to a test compound, and detecting binding between the adsorbentand the analyte by desorption spectrometry.

[0321] Rapid screening of combinatorial libraries for drug candidatesrequires the ability to expose target interactions to thousands of drugsand identify agents that interfere with or promote the interaction.Retentate chromatography enables one to dock one member of aligand/receptor pair to a substrate and to use it as a secondaryadsorbent. Then, after exposing the member to its partner and to theagent, one can determine by desorption spectrometry whether and to whatextent the partner has bound. Advantages of retentate chromatography inscreening methods include the ability to specifically dock the receptorto a substrate through an adsorbent, the ability to rapidly deploy thereceptor on many adsorbent spots for parallel processing, and the speedof throughput that is possible by reading results through desorptionspectrometry.

[0322] 1. Screening Assay

[0323] The method involves providing an adsorbent; contacting theadsorbent with the target analyte in the presence and absence of theagent under one or more selectivity conditions and determining whetherthe amount of binding with and without the agent. The amount of bindingis determined by retentate chromatography (e.g., by preparing arecognition profile). The experiment can be carried out with a controlin which no agent is added, or a control in which a different amount ortype of agent is added and the zero amount is determined byextrapolation. A statistically significant difference in the amount ofbinding (p<0.05) indicates that the test agent modulates binding.

[0324] This method is particularly useful to screen analytes (e.g.,proteins) as drug target candidates. After development of the proteinretention map or recognition profile from serum or some other targetcell type, the agent is exposed to the array of retained analyte attheir addressable locations. After binding is allowed, unbound agent iseluted or washed away. Those analytes that retained bound agent underthe selectivity conditions specified are identified directly bydesorption mass spectrometry, because the agent itself appears as a newcomponent in the retention map (i.e, the agent is desorbed and detecteddirectly). This method is particularly useful to screen drug candidates,both agonists and antagonists, for their ability to bind analytes ormodulate one or more biological processes.

[0325] 2. Receptor and Ligand

[0326] The adsorbent and the target analyte need not engage in specificbinding. However, in particularly useful methods the adsorbent and thetarget analyte are a ligand/receptor pair.

[0327] In one embodiment, the ligand/receptor pair are a hormone and acell surface receptor or an intracellular receptor. The adsorbent can bean entire cell or cell membrane in the case of a membrane-boundreceptor. A protein receptor or other drug target candidate may be usedas an adsorbent to screen combinatorial drug libraries. Hundreds orthousands of drug candidates can be applied to a single receptor type oraddressable location. After removal of unbound and weakly bound drugcandidates (i.e., agents) the bound agents are detected and identifiedby desorption spectrometry.

[0328] In another embodiment, the adsorbent is an enzyme that binds andmodifies the target substrate. The agents are screened for their abilityto modulate enzymatic transformation of the analyte. For example,enzymatic activity can be detected because the recognition profile of ananalyte may differ from that of the product of enzyme activity.Differential retention indicates that the agent alters binding.

[0329] The receptor/receptor can be retained on the substrate in avariety of ways. In one method, the receptor/ligand is directly retainedby a non-specific adsorbent. In another method, the adsorbent isspecific for the receptor/ligand. For example, the adsorbent can containan antibody specific for the receptor/ligand. The receptor/ligand can bea fusion protein in which the fusion moiety specifically binds theadsorbent, for example, in the manner that an Fc fragment binds proteinA. In one method, a genetic package, such as a phage from a phagedisplay library, that has on its surface a polypeptide that specificallybinds the receptor/ligand, is bound to the substrate. The ligand iscaptured by the polypeptide. Also, the adsorbent can be an analytealready docked to the substrate, i.e., it can be a secondary adsorbent,a tertiary adsorbent, etc.

[0330] This invention provides a particularly useful method to evaluateboth the direct and indirect consequences of drug (or other agent)binding to a target. The detection of one or more analytes in aretentate map generated from the proteins of a target cell type may bealtered due to the action of the agent (e.g., drug candidate) on 1) thetarget binding protein itself, 2) some other analyte (not the drugbinding protein), or 3) on gene expression (up or down regulation). Itis the high resolving and information generating power of retentatechromatography to detect these changes, i.e., drug induced differencesin the generic retentate map or recognition profile observed with andwithout drug, that makes this method one of the most powerful toolsavailable for proteomics, functional genomics, drug discovery,therapeutic drug monitoring, and clinical diagnostics.

[0331] 3. Test Agents

[0332] A test agent that is to be screened for its ability to modulateprothymosin expression is administered to the test animal or to thecultured cells in vitro. The choice of the agent to be tested is left tothe discretion of the practitioner. However, because of their varietyand ease of administration as pharmaceuticals, small molecules arepreferred as test agents.

[0333] a. Chemistry

[0334] The agent to be tested can be selected from a number of sources.For example, combinatorial libraries of molecules are available forscreening. Using such libraries, thousands of molecules can be screenedfor regulatory activity. In one preferred embodiment, high throughputscreening methods involve providing a library containing a large numberof potential therapeutic compounds (candidate compounds). Such“combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

[0335] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26,1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993),random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33,isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanonesU.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepines5,288,514, and the like).

[0336] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).

[0337] L. Methods for Generating Agents that Specifically Bind anAnalyte

[0338] This invention provides methods for generating agents, e.g.,single chain antibodies, that specifically bind to a target analyte.These agents are useful, e.g., as specific diagnostic agents for dockingtargets in the study of ligand/receptor interactions. The method isparticularly useful for generating agents against targets that may onlybe isolated in such small quantities that it is not possible orpractical to generate antibodies by immunizing an animal. The methodinvolves the steps of providing a substrate having a target attachedthereto; providing a display library of genetic packages that displayagents to be screened; exposing the library to the target tospecifically retain genetic packages through interaction with the targetand detecting retained genetic packages by desorption spectrometry.

[0339] These steps can be conducted in parallel for a large number ofadsorbent-analyte candidates within complex populations without transferlosses and ambiguities associated with separate selection and detectionprocedures, including off-line amplification and labeling strategiesassociated with indirect detection means.

[0340] 1. Providing the Substrate

[0341] The first step of the method involves providing a substrate thatcomprises an adsorbent that will serve as a target for a polypeptideagent of a display library to be screened. In one embodiment, thesubstrate is provided with the target adsorbent already attached. Inanother embodiment, the substrate is provided by providing a substratethat has an adsorbent that binds a target analyte, exposing theadsorbent to the analyte under elution conditions to allow retention ofthe analyte, and using the target adsorbent as the target for thedisplay library. In one embodiment, the target is differentiallyexpressed between two cell types that are being compared. For example,the targets may be derived from differentially expressed mRNA or may bedifferentially expressed polypeptides. Methods of identifying suchdifferentially expressed proteins by retentate chromatography methodsare described above.

[0342] Once a differentially expressed protein analyte is identified,one can develop a selectivity condition that unambiguously resolves theanalyte. More preferably, retention of the analyte is specific orexclusive. The methods for progressive resolution of analytes describedabove make it possible to identify selectivity conditions thatspecifically bind a target analyte from a complex sample. In oneembodiment, the bound target can be modified, e.g., by exposure to anenzyme.

[0343] Alternatively, the method can begin at the mRNA or EST stage. Inthis method, differentially expressed mRNAs or ESTs are identified byroutine methods. Then, these molecules are transcribed and translated invitro and in situ on an adsorbent for docking. For example, a substratefor desorption spectrometry having a plurality of adsorbent spots isprepared. The substrate is overlaid with a cylindrical tube, therebycreating a well with the adsorbent at the base of the well. In the wellone places reagents for in vitro transcription and translation of thedifferentially expressed mRNA (usually in the form of cDNA). (Formethods see, e.g., Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.)) Translation of the mRNAor EST produces a polypeptide that is adsorbed. The cylindrical tube isremoved and the adsorbent spots are washed with an eluant, so as toidentify a selectivity condition that retains the polypeptide analyte.

[0344] 2. Providing the Display Library

[0345] The second step involves providing a display library. The displaylibrary is comprised of genetic packages that display on their surfacesany sort of combinatorial library of peptides (“polypeptide agents”).However, single chain antibodies are attractive because they can be usedin subsequent immunoassays.

[0346] Many kinds of display libraries and their uses are known in theart. A basic concept of display methods is the establishment of aphysical association between a polypeptide ligand to be screened and arecoverable polynucleotide that encodes the polypeptide. This physicalassociation is provided by a multimeric molecular complex, in this casethe genetic package, e.g., the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypeptides and their genetic material allows simultaneous massscreening of very large numbers of genetic packages bearing differentpolypeptides. Genetic packages displaying a polypeptide with affinity toa target bind to the target and these packages are enriched by affinityscreening to the target. The identity of polypeptides displayed fromthese packages can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.

[0347] The genetic packages most frequently used for display librariesare bacteriophage, particularly filamentous phage, and especially phageM13, Fd and F1. Most work has inserted libraries encoding polypeptidesto be displayed into either gIII or gVIII of these phage forming afusion protein. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989;MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO92/18619 (gene VIII). See, also Cwirla et al., Proc. Natl. Acad. Sci.USA 87, 6378-6382 (1990); Devlin et al., Science 249, 404-406 (1990),Scott & Smith, Science 249, 386-388 (1990); Ladner et al., U.S. Pat. No.5,223,409 and Ladner et al. U.S. Pat. No. 5,571,698. Such a fusionprotein comprises a signal sequence, usually from a secreted proteinother than the phage coat protein, a polypeptide to be displayed andeither the gene III or gene VIII protein or a fragment thereof.Exogenous coding sequences are often inserted at or near the N-terminusof gene III or gene VIII although other insertion sites are possible.Some filamentous phage vectors have been engineered to produce a secondcopy of either gene III or gene VIII. In such vectors, exogenoussequences are inserted into only one of the two copies. Expression ofthe other copy effectively dilutes the proportion of fusion proteinincorporated into phage particles and can be advantageous in reducingselection against polypeptides deleterious to phage growth. Display ofantibody fragments on the surface of viruses which infect bacteria(bacteriophage or phage) makes it possible to produce human sFvs with awide range of affinities and kinetic characteristics.

[0348] In another variation, exogenous polypeptide sequences are clonedinto phagemid vectors which encode a phage coat protein and phagepackaging sequences but which are not capable of replication. Phagemidsare transfected into cells and packaged by infection with helper phage.Use of phagemid system also has the effect of diluting fusion proteinsformed from coat protein and displayed polypeptide with wild-type copiesof coat protein expressed from the helper phage. See, e.g., Garrard, WO92/09690.

[0349] Eukaryotic viruses can be used to display polypeptides in ananalogous manner. For example, display of human heregulin fused to gp70of Moloney murine leukemia virus has been reported by Han et al., Proc.Natl. Acad. Sci. USA 92, 9747-9751 (1995). Spores can also be used asreplicable genetic packages. In this case, polypeptides are displayedfrom the outer surface of the spore. For example, spores from B.subtilis have been reported to be suitable. Sequences of coat proteinsof these spores are provided by Donovan et al., J. Mol. Biol. 196, 1-10(1987).

[0350] Cells can also be used as replicable genetic packages.Polypeptides to be displayed are inserted into a gene encoding a cellprotein that is expressed on the cells surface. Bacterial cellsincluding Salmonella typhimurium, Bacillus subtilis, Pseudomonasaeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseriagonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxellabovis, and especially Escherichia coli are preferred. Details ofouter-surface proteins are discussed by Ladner et al., U.S. Pat. No.5,571,698, and Georgiou et al., Nature Biotechnology 15, 29-34 (1997)and references cited therein. For example, the lamB protein of E. coliis suitable.

[0351] 3. Screening the Display Library

[0352] The third step involves screening the display library to identifya ligand that specifically binds the target. The substrate bearing thetarget is exposed to the display library that displays polypeptideagents under elution conditions appropriate for specific binding betweenthe polypeptide and a target molecule. Genetic packages that have agentsthat recognize the target bind to the target already attached to thesubstrate. Removing unbound particles and retention of bound particlesresults from exposure to the elution condition.

[0353] A population of genetic packages, in this case M13 phage,representing approximately 10¹¹ plaque forming units (pfu) per mL areintroduced to a substrate with an addressable array of bound targetadsorbents (e.g., protein). Upon contact, a selectivity conditionoptimized for the target adsorbent (i.e., selectivity threshold modifieror eluent) is chosen, such that only a small subset of total phagegenotypes are selectively retained, preferably fewer than 5-10. Notethat unbound phage (i.e., phage not bound to the target adsorbent) andphage loosely bound to the target adsorbent are eliminated by exposureto eluents that disrupt all but the most selective analyte-targetadsorbent interaction(s). Those phage displaying polypeptides with thehighest affinity for the target adsorbent are selectively retained.

[0354] 4. Detecting Bound Genetic Packages Containing Agents thatSpecifically Bind the Target

[0355] In the fourth step, the binding of genetic packages to the targetis detected by desorption spectrometry. For example, the M13 phage hasthousands of copies of a single coat protein. Upon striking the phagewith a laser in desorption spectrometry, the coat proteins becomedislodged and are detectable. In this way, one can determine whether thelibrary contained a phage having an agent that bound to the target. Inorder to have genetic packages for subsequent analysis, the screeningstep can be performed in parallel at different locations on a probe, orthe substrate can have a physical dimension sufficiently large so thatthe laser does not dislodge all the genetic packages bound to thesurface. This method is particularly powerful, because even a few phagebound to the analyte can be detected.

[0356] In the case of M13, the preferred detection method is to monitor,by desorption spectrometry, the appearance of the gene VIII coat proteinas a “marker” protein signal. In this manner, we have detected“positive” target adsorbents with as few as 5 phage particles (pfu)bound (phage particle number estimated by calculation from knowndilutions). Other phage markers, in order of preference, include gene V,gene X, and gene III (including their fusion products).

[0357] After detection of those adsorbent locations with the highestaffinity adsorbents, that is, those locations within the array with thefewest phage retained after exposure to high selectivity conditions(i.e., stringent eluents), the bound package can now be used as ajump-off point for other uses.

[0358] 5. Isolating the Genetic Package

[0359] In one embodiment, the method further involves isolating thegenetic package for further analysis. This analysis can involvereproducing the genetic package and isolating the polynucleotide fromit. The isolated phage are reproduced by the usual methods. For example,the retained phage can be exposed to a biological amplification vehicle,for example, E. coli, plus nutritive media to grow the genetic packagesfor subsequent analysis. Single clones can be further tested for abilityto bind to analyte retained on substrate.

[0360] 6. Sequencing the Nucleotide Sequence Encoding the PolypeptideAgent

[0361] Sequencing the nucleotide sequence encoding the polypeptide agentof a bound genetic package provides information for producing thepolypeptide agent. Sequencing can involve isolating the genetic packagefrom the adsorbent, reproducing it, isolating the polynucleotide, andsequencing the nucleotide sequence by any available means. In anothermethod, the genetic packages can be reproduced in situ by contacting thesubstrate with appropriate materials, such as cells subject to infectionby the genetic package. In another embodiment, sequencing is performedin situ. The method can involve lysing the genetic packages andamplifying the nucleotide sequence by any known means, e.g., PCR.Several different genetic packages may have bound to different epitopesavailable on the surface. In this case, one may alter the elutionconditions so that only one kind of phage binds to an epitope.

[0362] 7. Producing the Polypeptide Agent

[0363] One valuable next step involves producing the polypeptide agent.The isolated agent can be used, e.g., as an adsorbent for specificdetection of the target in diagnostics or for the study ofligand/receptor interactions.

[0364] In one method, producing the polypeptide involves firstsequencing the nucleotide sequence that encodes the polypeptide. Theamino acid sequence can be derived from the nucleotide sequence.Sequencing can be accomplished by the method as described above. Thesequence can be the basis for recombinant or chemical synthesis of thepolypeptide agent.

[0365] In another method, the polypeptide can be produced by reproducingthe genetic package. This is particularly effective when the geneticpackage contains many copies of the polypeptide agent. The geneticpackage can be reproduced in situ or after isolation.

[0366] A method of producing the polypeptide recombinantly can proceedas follows. The nucleotide sequence encoding the polypeptide is eithersequenced or isolated by any means such as those discussed. Then, thenucleotide sequence is included in an expression vector. The expressionvector contains an expression control sequence operatively linked to thenucleotide sequence encoding the polypeptide. The expression vector canthen be used to express the polypeptide agent recombinantly by meanswell known in the art.

[0367] It is understood that the target can contain more than oneepitope. Accordingly, the method can produce more than one polypeptideagent specific for the target.

[0368] Target-specific agents can then be used as adsorbents for probesused in clinical diagnostics or drug discovery. That is, because suchprobes contain on their surface agents that specifically bind thetarget, they can be used to isolate the target from complex mixtures,such as biological samples, and to detect the target by desorptionspectrometry. Furthermore, because the interaction between the agent andthe target can be biospecific, it is likely to involve a greateraffinity between the two than an adsorbent developed by the progressiveresolution method, described above.

[0369] 8. Isolating Peptide Epitopes of a Target

[0370] In one version this method allows one to isolate peptide epitopesof a target analyte. The method employs an “anti-idiotypic”-likeapproach. In summary, the epitopes of a target analyte are screenedwith, e.g., a phage display library. The isolated phage contain, e.g.,single chain antibodies that recognize the epitopes of the analyte.These phage are used, in turn, to screen a second display library. Thephage from the second library that bind to the single chain antibodiesof the first contain displayed polypeptides that mimic the structure ofthe epitope recognized by the single chain antibodies.

[0371] In one embodiment of this method, a nucleotide sequence encodinga polypeptide agent that binds the target analyte is used to produce M13phage in which the agent is displayed as a fusion with gene VIII. Thus,this phage has a coat with hundreds of copies of the target peptide onits surface. This phage is then docked to the adsorbent. Docking can beaccomplished through, e.g., a ligand that binds gene III, or gene IIIcan be modified to include a receptor for a ligand on the substrate. Thephage is then exposed to a second display library. Genetic packages fromthe library that bind to the docked phage are detected and isolated asdescribed. Preferably, the second display library contains a mass labelof some sort so that their gene VIII protein can be distinguished fromgene VIII of the phage docked to the substrate. Thus, the identificationof a substance as an “analyte target” or as an adsorbent can depend uponwhether the bound substance is used, subsequently, to bind anothersubstance. As one can see, the ability to bind a substance to an alreadydocked substance can continue, as can methods of identifying conditionsthat selectively remove the terminally bonded substance.

EXAMPLES

[0372] The following examples are offered by way of illustration, not byway of limitation.

[0373] In the following examples, the following products and terms areemployed. Chicken egg white lysozyme (1 μl diluted to 10 picomole/μlwater), is available from Sigma Chemical Company, St. Louis, Mo. “Humanserum” refers to a composition of 1 μl of human serum diluted 1 to 5 in20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

[0374] As used herein, “mg” means milligram(s); “ml” meansmilliliter(s); “μl” means microliter(s); “cm” means centimeter(s); “nm”means nanometer(s); “M” means molar; “mM” means millimolar; “min” meansminute(s); “%” or “percent” is percent by weight unless otherwisespecified; “NaCl” means sodium chloride; “TFA” means trifluoroaceticacid.

[0375] I. Protocols for Retentate Chromatography

[0376] The following protocols are examples of procedures for performingretentate chromatography.

[0377] A. Protocol for Retentate Mapping (Using Chromatographic SeriesArray)

[0378] 1. Sample Treatment

[0379] Dilute the biological sample (e.g., serum, urine, cell extract orcell culture medium) in 0.01% Triton X100 in HEPES or 20 mM Naphosphate, pH 7.2. Centrifuge to clarify sample if necessary.

[0380] 2. Sample Application

[0381] Add sample (1-5 μl) to a spot of Anionic, Normal phase orTED-Cu(II) adsorbent array. For a hydrophobic adsorbent array preweteach spot with 0.5 μl acetonitrile containing 0.5% TFA. Add sample tothe spot before the acetonitrile is dry. Allow sample to concentrate(almost to dryness) on the spot.

[0382] 3. Washing

[0383] a. Anionic Adsorbent Array

[0384] Wash spot 1 with 20 mM HEPES or Na phosphate, pH 7.2. Add thefirst 2 μl of wash solution to the spot before the sample is completelydry. Let the wash solution sit on the spot for at least 15 sec. Pipetout and in 10 times. Remove the first wash completely, repeat with thesecond wash of 2 μl of the solution.

[0385] Wash spot 2 with 0.2 M NaCl in 20 mM Na phosphate, pH 7.2 asabove.

[0386] Wash spot 3 with 1 M NaCl in 20 mM Na phosphate, pH 7.2 as above.

[0387] Wash spot 4 with 20 mM TrisHCl, pH 8.5 as above.

[0388] Wash spot 5 with 0.1 M Na acetate, pH 4.5 as above.

[0389] Wash spot 6 with 0.05% Triton X100 in 20 mM HEPES or Naphosphate, pH 7.2 as above.

[0390] Wash spot 7 with 3 M urea in 20 mM HEPES or Na phosphate, pH 7.2as above.

[0391] Wash spot 8 with 10% acetonitrile in water as above.

[0392] Wash the whole array with water thoroughly.

[0393] Air dry the chip.

[0394] Add 0.3 μl Energy Absorbing Molecule (saturated solution preparedin 50% acetonitrile, 0.5% trifluoroacetic acid).

[0395] Air dry the chip.

[0396] Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

[0397] b. Normal Phase Adsorbent Array

[0398] Wash spot 1 with 5 mM HEPES, pH 7. Add the first 2 μl of washsolution to the spot before the sample is completely dry. Let the washsolution sit on the spot for at least 15 sec. Pipet out and in 10 times.Remove the first wash completely, repeat with the second wash of 2 μl ofthe solution.

[0399] Wash spot 2 with 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 asabove.

[0400] Wash spot 3 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2 as above.

[0401] Wash spot 4 with 0.1 M Na acetate, pH 4.0 as above.

[0402] Wash spot 5 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 MNaCl, pH 7.2 as above.

[0403] Wash spot 6 with 3 M urea in 20 mM Na phosphate, 0.15 M NaCl, pH7.2 as above.

[0404] Wash spot 7 with 1% TFA as above.

[0405] Wash spot 8 with 30% isopropanol:acetonitrile (1:2) in water asabove.

[0406] Wash the whole array with water thoroughly.

[0407] Air dry the chip.

[0408] Add 0.3 μl Energy Absorbing Molecule (saturated solution preparedin 50% acetonitrile, 0.5% trifluoroacetic acid).

[0409] Air dry the chip.

[0410] Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

[0411] c. TED-Cu(II) Adsorbent Array

[0412] Wash spot 1 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2. Add thefirst 2 μl of wash solution to the spot before the sample is completelydry. Let the wash solution sit on the spot for at least 15 sec. Pipetout and in 10 times. Remove the first wash completely, repeat with thesecond wash of 2 μl of the solution.

[0413] Wash spot 2 with 20 mM imidazole in 20 mM Na phosphate, 0.5 MNaCl, pH 7.2 as above.

[0414] Wash spot 3 with 100 mM imidazole in 20 mM Na phosphate, 0.5 MNaCl, pH 7.2 as above.

[0415] Wash spot 4 with 0.1 M Na acetate, 0.5 M NaCl, pH 4.0 as above.

[0416] Wash spot 5 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 MNaCl, pH 7.2 as above.

[0417] Wash spot 6 with 3 M urea in 20 mM Na phosphate, 0.15 M NaCl, pH7.2 as above.

[0418] Wash spot 7 with 1% TFA as above.

[0419] Wash spot 8 with 10% acetonitrile in water as above.

[0420] Wash the whole array with water thoroughly.

[0421] Air dry the chip.

[0422] Add 0.3 μl Energy Absorbing Molecule (saturated solution preparedin 50% acetonitrile, 0.5% trifluoroacetic acid).

[0423] Air dry the chip.

[0424] Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

[0425] d. Hydrophobic Adsorbent Array

[0426] Wash spot 1 with 5% acetonitrile in 0.1% TFA. Add the first 2 μlof wash solution to the spot before the sample is completely dry. Letthe wash solution sit on the spot for at least 15 sec. Pipet out and in10 times. Remove the first wash completely, repeat with the second washof 2 μl of the solution.

[0427] Wash spot 2 with 50% acetonitrile in 0.1% TFA as above.

[0428] Wash spot 3 with 0.05% Triton X100 in 20 mM Na phosphate, 0.15 MNaCl, pH 7.2 as above.

[0429] Wash spot 4 with 3M urea in 20 mM Na phosphate, 0.15 M NaCl, pH7.2 as above.

[0430] Wash the whole array with water thoroughly.

[0431] Air dry the chip.

[0432] Add 0.3 μl Energy Absorbing Molecule (saturated solution preparedin 50% acetonitrile, 0.5% trifluoroacetic acid).

[0433] Air dry the chip.

[0434] Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

[0435] B. Protocol for Antibody-Antigen Assay; Receptor-Ligand Assay(Using Pre-activated Adsorbent Array)

[0436] 1. Immobilization of Antibody on Pre-activated Adsorbent Array

[0437] Place a pre-activated adsorbent array on a flat clean surface.Spot the antibody or receptor or control solution onto each spot of apre-activated adsorbent array prewetted with 0.5 μl of isopropanol (add1 μl antibody/spot before the isopropanol is dry).

[0438] Incubate (4° C. or room temperature, 2-18 h) in a humid chamber.

[0439] Use pipet to remove remaining solution from the spots.

[0440] Block residual active sites on the spots by adding 1 ml of 1 Methanolamine, pH 7.4 in PBS over the entire chip and incubate in a humidchamber (room temperature 30 min).

[0441] Wash the chip twice with 1% Triton X-100 in PBS. Submerge thechip in about 9 ml of wash solution in a 15 ml conical plastic tube androck on benchtop agitator for at least 15 minutes.

[0442] Wash with 0.5 M NaCl in 0.1 M sodium acetate, pH 4.0 as above.

[0443] Wash with 0.5 M NaCl in 0.1 M TrisHCl, pH 8.0 as above.

[0444] Rinse with PBS as above. Then cover the chip with PBS and storeat 4° C. until ready to use.

[0445] 2. Binding of Antigen or Ligand

[0446] Gently shake or blot off PBS on the Chip.

[0447] Add 1-5 μl of sample to each spot. For samples with very lowantigen or ligand concentration, put the adsorbent array into abioprocessor. Wash the spots on the chip and Bioprocessor wells with 200al PBS two times. Add up to 300 μl of sample to each well.

[0448] Seal with adhesive tape.

[0449] Incubate with shaking (4° C. or room temperature, 1-18 h).

[0450] 3. Washing

[0451] Remove sample from the spots, wash each spot with 2 μl of 0.1%Triton X100 in PBS, pH 7.2, two times. Add the first 2 μl of washsolution to the spot. Let the wash solution sit on the spot for at least15 sec. Pipet out and in 10 times. Remove the first wash completely,repeat with the second wash of 2 μl of the solution. This is followed bya wash with 0.5 M NaCl in 0.1 M HEPES, pH 7.4.

[0452] Wash the whole array with water thoroughly.

[0453] 4. Analysis of Retained Proteins

[0454] Air dry the chip.

[0455] Add 0.3 μl Energy Absorbing Molecule (saturated solution ofSinapinic Acid or EAM1 or

[0456] CHCA prepared in 50% acetonitrile, 0.5% trifluoroacetic acid).

[0457] Air dry the chip.

[0458] Analyze the retained protein on each spot with laserdesorption/ionization time-of-flight mass spectrometer.

[0459] II. Recognition Profile of Lysozyme

[0460] We generated a recognition profile for lysozyme usinghigh-information resolution retentate chromatography. The profileincludes resolution of lysozyme with six adsorbents, each under avariety of different selectivity threshold modifiers. The result is 40different spectrographs that differently characterize thephysico-chemical properties of lysozyme.

[0461] A. Lysozyme Recognition Profile Using a Hydrophilic AdsorbentArray

[0462] Chicken egg white lysozyme is added to various spots of achromatographic series adsorbent array of a silicon oxide adsorbent on astainless steel substrate. After incubation in a moist chamber at roomtemperature for 15 min., each different spot of adsorbent is washed withone of the following eluants (selectivity threshold modifiers):

[0463] (1) 20 mM sodium phosphate buffer, pH 7.0,

[0464] (2) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0465] (3) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0466] (4) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

[0467] (5) 1% TFA,

[0468] (6) 10% acetonitrile in water,

[0469] (7) 20% acetonitrile in water,

[0470] (8) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, and

[0471] (9) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

[0472] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer, using a nitrogen laser(355 nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c (available from Galactic Industries Corporation)for data overlay presentation.

[0473]FIG. 5A shows the composite mass spectrum of a lysozymerecognition profile on a normal phase chromatographic series adsorbentarray. The bottom profile shows the lysozyme signal intensity retainedon the silicon oxide adsorbent after washing with pH 7 buffer alone.Inclusion of sodium chloride (0.2-0.4 M) in the selectivity thresholdmodifier decreases the retention of lysozyme. This indicates that theinteraction of lysozyme (a basic protein) with a silicon oxide(negatively charged at pH 7) adsorbent involves an ion exchangemechanism. Lowering the pH of the selectivity threshold modifier, forexample to pH 4.5 in the sodium acetate buffer, or <2 in 1% TFA, almostcompletely eliminates the negative charge on the silicon oxideadsorbent, and lysozyme is not retained any longer. Including polaritymodulating agents, (e.g., organic solvents (e.g., acetonitrile), ordetergent (e.g., Tween20), or urea in the selectivity threshold modifieralso reduces the interaction of lysozyme with the silicon oxideadsorbent. This indicates that the other interaction mechanism involvesa hydrophilic interaction.

[0474] B. Lysozyme Recognition Profile Using a Hydrophobic AdsorbentArray

[0475] Chicken egg white lysozyme is added to various spots of achromatographic series adsorbent array of polypropylene (C₃ hydrophobic)adsorbent coated on silicon oxide-coated stainless steel substrate.After incubation in a moist chamber at room temperature for 15 min.,each different spot of adsorbent is washed with one of the followingeluants (selectivity threshold modifiers):

[0476] (1) 0.1% TFA,

[0477] (2) 10% acetonitrile in 0.1% TFA,

[0478] (3) 20% acetonitrile in 0.1% TFA,

[0479] (4) 50% acetonitrile in 0.1% TFA,

[0480] (5) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, and

[0481] (6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH7.0.

[0482] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Afterwards, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0483]FIG. 5B shows the composite mass spectrum of lysozyme recognitionprofile on a hydrophobic C₃ chromatographic series adsorbent array. Thebottom profile shows the lysozyme signal intensity retained on thehydrophobic C₃ adsorbent after washing with 0.1% TFA alone. Including apolarity modulating agent, (e.g., acetonitrile) in the selectivitythreshold modifier decreases the retention of lysozyme on thehydrophobic C₃ adsorbent. The acetonitrile concentration range forelution of lysozyme from the hydrophobic C₃ adsorbent is between 20-50%.Including detergent (Tween20), or urea, in the selectivity thresholdmodifier does not significantly reduce the retention of lysozyme on thehydrophobic C₃ adsorbent.

[0484] C. Lysozyme Recognition Profile Using a Phenyl HydrophobicAdsorbent Array

[0485] Chicken egg white lysozyme is added to various spots of anadsorbent array of polystyrene (phenyl hydrophobic) adsorbent coated onsilicon oxide-coated stainless steel substrate. After incubation in amoist chamber at room temperature for 15 min., one spot of adsorbent iswashed with one of the following eluants (selectivity thresholdmodifiers):

[0486] (1) 0.1% TFA,

[0487] (2) 10% acetonitrile in 0.1% TFA,

[0488] (3) 20% acetonitrile in 0.1% TFA,

[0489] (4) 50% acetonitrile in 0.1% TFA,

[0490] (5) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, and

[0491] (6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH7.0.

[0492] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0493]FIG. 5C shows the composite mass spectrum of the lysozymerecognition profile on the hydrophobic phenyl chromatographic seriesadsorbent array. The bottom profile shows the lysozyme signal intensityretained on the hydrophobic phenyl adsorbent after washing with 0.1% TFAalone. Including a polarity modulating agent, (e.g., acetonitrile) inthe selectivity threshold modifier decreases the retention of lysozyme.The acetonitrile concentration range for elution of lysozyme from thehydrophobic C₃ adsorbent is between 20-50%, however, when the lysozymepeak intensities retained on the C₃ and phenyl surface are comparedunder the same 20% acetonitrile wash condition, the interaction oflysozyme with the phenyl adsorbent is less strong. Including detergent(e.g., Tween20), or urea, in the selectivity threshold modifier alsosignificantly reduces the retention of lysozyme on the hydrophobicphenyl adsorbent.

[0494] D. Lysozyme Recognition Profile Using an Anionic Adsorbent Array

[0495] Chicken egg white lysozyme is added to various spots of anadsorbent array of anionic group (SO₃ ⁻) adsorbent (i.e., a cationicexchange adsorbent) coated on silicon oxide-coated stainless steelsubstrate. After incubation in a moist chamber at room temperature for15 min., each different spot of adsorbent is washed with one of thefollowing eluants (selectivity threshold modifiers):

[0496] (1) 20 mM sodium phosphate buffer, pH 7.0,

[0497] (2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0498] (3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0499] (4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0500] (5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

[0501] (6) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, and

[0502] (7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

[0503] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0504]FIG. 5D shows the composite mass spectrum of the lysozymerecognition profile on a cation exchange chromatographic series array.The bottom profile shows the lysozyme signal intensity retained on theanionic adsorbent after washing with pH 7 buffer alone. Includingincreasing concentrations of sodium chloride (0.1-0.4 M) in theselectivity threshold modifier decreases the retention of lysozyme. Thisindicates that the interaction of lysozyme (a basic protein) with theanionic adsorbent involves an ion exchange mechanism. A 0.4 M NaClconcentration is required to elute the lysozyme. Lowering the pH of theselectivity threshold modifier to pH 4.5 in the sodium acetate buffer,does not affect the retention of lysozyme on a strong anionic adsorbent.Including a polarity modulating agent (e.g., a detergent such asTween20, or urea) in the selectivity threshold modifier reduces theinteraction of lysozyme with an anionic adsorbent. This indicates thatthe interaction of a hydrophobic lysozyme protein with the anionicadsorbent is modulated by the polarity of the eluant.

[0505] E. Lysozyme Recognition Profile Using an Cationic Adsorbent Array

[0506] Chicken egg white lysozyme is added to various spots of anadsorbent array of cationic (quaternary amine) adsorbent coated onsilicon oxide-coated stainless steel substrate. After incubation in amoist chamber at room temperature for 15 min., each different spot ofadsorbent is washed with one of the following eluants (selectivitythreshold modifiers):

[0507] (1) 20 mM sodium phosphate buffer, pH 7.0,

[0508] (2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0509] (3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0510] (4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

[0511] (5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

[0512] (6) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, or

[0513] (7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0.

[0514] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0515]FIG. 5E shows the composite mass spectrum of the lysozymerecognition profile on the cationic (anion exchange) adsorbentchromatographic series adsorbent array. The retention of the basiclysozyme protein on the cationic adsorbent is very weak. The effect ofmodulating the selectivity threshold modifiers on lysozyme retention isminimal.

[0516] F. Lysozyme Recognition Profile Using an Immobilized Metal IonAdsorbent Array

[0517] Chicken egg white lysozyme is added to various spots of anadsorbent array of immobilized metal (iminodiacetate-Cu) adsorbentcoated on silicon oxide-coated stainless steel substrate. Afterincubation in a moist chamber at room temperature for 15 min., eachdifferent spot of adsorbent is washed with one of the following eluants(selectivity threshold modifiers):

[0518] (1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

[0519] (2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl,pH 7.0,

[0520] (3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

[0521] (4) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, or

[0522] (5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

[0523] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0524]FIG. 5F shows the composite mass spectrum of the lysozymerecognition profile on the immobilized metal chromatographic seriesadsorbent array. The bottom profile shows the lysozyme signal intensityretained on the immobilized copper ion adsorbent after washing with pH 7buffer alone. Including a histidine-binding competitive affinity ligand(e.g., imidazole) in the selectivity threshold modifier eliminates theretention of lysozyme. This indicates that the interaction of lysozyme(which has a single histidine residue in the sequence) with animmobilized copper ion adsorbent involves a coordinate covalent bindingmechanism. Lowering the pH of the selectivity threshold modifier to pH4.5 in the sodium acetate buffer, also decreases the retention oflysozyme on the immobilized copper adsorbent. It is believed that thisis a result of the protonation of the histidine residue on lysozyme,which inhibits the coordinate covalent interaction. Including detergent(i.e., Tween20) does not affect the interaction. Including ureacompletely eliminates the retention of lysozyme on the immobilizedcopper adsorbent.

[0525] III. Resolution of Analytes in Human Serum

[0526] We resolved analytes in human serum using a variety of adsorbentsand eluants. These results show that analytes are differentiallyretained by different adsorbents, and that retention chromatography isable to provide information at both low and high molecular masses.

[0527] A. Human Serum Protein Recognition Profile Using an ImmobilizedMetal Ion Adsorbent Array

[0528] Human serum is added to various spots of an adsorbent array ofimmobilized metal ion (tris(carboxymethyl)ethylenediamine-Cu) adsorbentcoated on silicon oxide-coated stainless steel substrate. Afterincubation in a moist chamber at room temperature for 15 min., eachdifferent spot of adsorbent is washed with one of the following eluants(selectivity threshold modifiers):

[0529] (1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

[0530] (2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl,pH 7.0,

[0531] (3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

[0532] (4) 0.05% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0, and

[0533] (5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

[0534] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0535]FIGS. 6A and 6B show the composite mass spectrum at low and highmolecular mass of the serum protein recognition profile on theimmobilized metal chromatographic series adsorbent array. The bottomprofile shows the serum proteins retained on the immobilized copperadsorbent after washing with pH 7 buffer alone. Including ahistidine-binding competitive affinity ligand (e.g., imidazole), ordetergent (e.g., Tween20), or urea in the selectivity thresholdmodifier, or lowering the pH of the selectivity threshold modifier to4.5, differentially enhances or decreases the retention of differentcomponents of the complex protein mixture on the same adsorbent.

[0536] B. Human Serum Protein Recognition Profile Using a Plurality ofDifferent Adsorbents

[0537] Human serum is added to various spots of an adsorbent array ofthe following different adsorbents:

[0538] (1) C₃ hydrophobic,

[0539] (2) phenyl hydrophobic,

[0540] (3) anion exchange,

[0541] (4) cation exchange, and

[0542] (5) immobilized metal (tris(carboxymethyl)ethylenediamine-Cu).

[0543] Each adsorbent is coated on a silicon oxide-coated stainlesssteel substrate. After incubation in a moist chamber at room temperaturefor 15 min., each spot of adsorbent is washed with 0.05% Tween20 in 20mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 as the selectivitythreshold modifier.

[0544] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the mass spectrometer using a nitrogen laser (355nm) and a 60 cm flight tube. The data is analyzed by computer andexported to GRAMS/32c for data overlay presentation.

[0545]FIG. 7A and 7B show the composite mass spectrum of the serumprotein recognition profile on various adsorbents of a chromatographicseries adsorbent array. The use of a single selectivity thresholdmodifier on a plurality of different adsorbents (having differentbinding characteristics) differentially enhances or decreases theretention of different components of the complex protein mixture on thedifferent adsorbents.

[0546] IV. Resolution of Analytes in Preterm Infant Urine

[0547] We resolved analytes in preterm infant urine using a variety ofadsorbents and eluants. These results show that because adsorbentsretain analytes differentially, the use of various adsorbents providesgreat resolving ability. They also show the ability to identifyadsorbents that preferentially retain specific analytes, which is usefulfor developing purification schemes.

[0548] A. Resolution of Analytes in Preterm Infant Urine Using A Varietyof Adsorbents and the Same Eluant (Water)

[0549] Preterm infant urine (2 μl) is added to various spots of acarbonized PEEK polymer substrate coated with the following differentadsorbents:

[0550] (1) C₈ hydrophobic (Octyl Sepharose, available from Sigma),

[0551] (2) phenyl hydrophobic (Phenyl Sepharose, available from Sigma),

[0552] (3) anion exchange (Q Sepharose, available from Sigma),

[0553] (4) cation exchange (S Sepharose, available from Sigma),

[0554] (5) immobilized metal (IDA-Cu, Chelating Sepharose, availablefrom Pharmacia), and

[0555] (6) immobilized metal (tris(carboxymethyl)ethylenediamine-CuSepharose).

[0556] After incubation in a moist chamber at room temperature for 15min., each spot of adsorbent is washed with water as the selectivitythreshold modifier. Each wash includes pipetting 1 μl of wash solutionin and out of the spot of adsorbent three times. This is repeated with afresh aliquot of wash solution. An aliquot of 0.3 μl of sinapinic acid(5 mg/ml 50% acetonitrile:0.5% TFA) is added and allowed to air dry. Thearray is analyzed with the a laser desorption/ionization time-of-flightmass spectrometer from Hewlett Packard (Model 2030) that uses a nitrogenlaser (355 nm) and a 150 cm flight tube. The data is analyzed by HPMALDI TOF software and exported to GRAMS/32c for data overlaypresentation.

[0557]FIGS. 8A and 8B show the composite mass spectrum at low and highmolecular mass of the preterm infant urine protein recognition profileon the various adsorbents of a chromatographic series. The use of asingle selectivity threshold modifier (i.e., water) on the variousadsorbents (each having a different binding characteristic)differentially enhances or decreases the retention of differentcomponents of the complex protein mixture like on the differentadsorbents.

[0558] B. Resolution of Analytes in Preterm Infant Urine Using aHydrophobic Phenyl Adsorbent Indirectly Coupled to the Substrate andThree Different Eluants

[0559] Preterm infant urine (2 μl) is added to various spots of acarbonized PEEK polymer substrate coated with phenyl hydrophobicadsorbent (Phenyl Sepharose, available from Sigma). After incubation ina moist chamber at room temperature for 15 min., each spot of adsorbentis washed with one of the following eluants (selectivity thresholdmodifiers):

[0560] (1) water,

[0561] (2) 2M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH7.0, and

[0562] (3) 0.1% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0.

[0563] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This process is repeated with a freshaliquot of wash solution. Thereafter, the spot of adsorbent is washedwith 1 μl of water two times. An aliquot of 0.3 μl of sinapinic acid (5mg/ml 50% acetonitrile:0.5%TFA) is added and allowed to air dry. Thearray is analyzed with a laser desorption/ionization time-of-flight massspectrometer from Hewlett Packard (Model 2030) that uses a nitrogenlaser (355 nm) and a 150 cm flight tube. The data is analyzed by HPMALDI TOF software and exported to GRAMS/32c for data overlaypresentation.

[0564]FIG. 9 shows the composite mass spectrum of the preterm infanturine protein recognition profile on the hydrophobic phenyl adsorbent ofa chromatographic series. The application of various eluants havingdifferent elution characteristics on a single adsorbent differentiallyenhances or decreases the retention of different components of thecomplex protein mixture. One of the components (marked by *) isselectively retained on the hydrophobic phenyl adsorbent when 01%Tween20 in PBS is used as the eluant.

[0565] V. Identification of Proteins in Culture Medium from TwoDifferent Cell Lives

[0566] This example illustrates the identification of proteins that aredifferentially expressed in cells with adsorbent array: Chromatographicseries.

[0567] Two different breast cancer cell lines are cultured for the sameperiod of time in a constant composition culture medium. Afterconcentration with a filtration unit, an aliquot of 1 μl of each culturemedium is added to various spots of a an adsorbent array (CiphergenBiosystems, Inc., Palo Alto, Calif.) of immobilized metal(tris(carboxymethyl)ethylenediamine-Cu) adsorbent coated on siliconoxide-coated stainless steel as substrate. After incubation at roomtemperature in a moist chamber for 15 min., a spot of adsorbent iswashed with either one of the following eluants (selectivity thresholdmodifiers):

[0568] (1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

[0569] (2) 20 mM imidazole in 20 mM sodium phosphate buffer, 05 M NaCl,pH 7.0,

[0570] (3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

[0571] (4) 0.1% Tween20 in 20 mM sodium phosphate buffer, 0.15 M NaCl,pH 7.0,

[0572] (5) 3M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,or

[0573] (6) 1% TFA.

[0574] Each wash includes pipetting 1 μl of wash solution in and out ofthe spot of adsorbent three times. This is repeated with a fresh aliquotof wash solution. Afterwards, the spot of adsorbent is washed with 1 μlof water two times. An aliquot of 0.3 μl of sinapinic acid (5 mg/ml 50%acetonitrile:0.5% trifluoroacetic acid) is added and allowed to air dry.The array is analyzed with a laser desorption/ionization time-off-flightmass spectrometer that uses a nitrogen laser (355 nm) and a 60 cm flighttube. The data is analyzed by computer and exported to GRAMS/32c(Galactic Industries Corporation) for data overlay presentation.

[0575]FIG. 10A shows the composite mass spectrum of cell secretedprotein recognition profile of cell line 1 on an immobilized metal (Cu)chromatographic series adsorbent array. The application of variouseluants of different selectivity thresholds on a single adsorbentdifferentially enhances or decreases the retention of differentcomponents of a complex protein mixture like cell culture medium.

[0576]FIG. 10B shows the composite mass spectrum of cell secretedprotein recognition profiles of both cell lines on an immobilized metal(Cu) chromatographic series adsorbent array. The same eluant, 0.1%Tween20+3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,is used to wash away unretained materials. The peak marked 7532 Da isthe major retained peak in cell line 1 secreted protein that is notexpressed in cell line 2.

[0577]FIG. 10C shows the composite mass spectrum of cell secretedprotein recognition profiles of cell line 1 on an immobilized metal (Ni)chromatographic series adsorbent array. Using the same eluant, 0.1%Tween20+3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,but employing an adsorbent of different surface interaction potential(i.e., immobilized Ni metal vs immobilized Cu metal), the 7532 Da peakis the only retained protein among all the cell line 1 secretedproteins. The inset shows the same mass spectrum on an expanded scale.The smaller peak at 3766 Da is the doubly charged species of the sameprotein.

[0578]FIG. 10D shows the composite mass spectrum of cell secretedprotein recognition profiles of cell line 1 on an immobilized metal (Ni)chromatographic series adsorbent array before (lower profile) and after(top profile) in situ trypsin digestion. The peptide map generated for apure protein is a fingerprint of that protein and can be used foridentification.

[0579] VI. Comparison of Retentate Chromatography with 2D GelElectrophoresis

[0580] One advantage of retentate chromatography is the ability torapidly resolve analytes in a variety of dimensions, resulting in highinformation content about a variety of physico-chemical characteristics.In contrast, 2D gel electrophoresis provides resolution in twodimensions only.

[0581]FIG. 11 shows a preterm infant urine protein recognition profileon phenyl hydrophobic adsorbent of a chromatographic series. Theapplication of various eluants and adsorbents yields multi-dimensionalinformation. The use of different selectivity conditions differentiallyenhances or decreases the retention of various components of a complexprotein mixture (such as preterm infant urine), resulting in detailedresolution of analytes.

[0582] In contrast, FIG. 12 shows a two-dimensional separation ofproteins in preterm infant urine according to pI and molecular mass. Thegel provides information about two dimensions, only, as compared to thesix dimensions used as adsorbents in retentate chromatography. Spots arenot as well resolved as by mass spectrometry and resolution at very highand very low molecular masses is limited.

[0583] VII. Sequential Extraction of Analytes from a Sample

[0584] Analytes can be sequentially extracted from a sample by seriallyexposing the sample to a selectivity condition followed by collection ofthe un-retained sample.

[0585] A Hemophilus knockout mutant lysate was prepared in 10% glycerol50 mM EDTA. After centrifugation, the supernate was diluted 1:3 in 0.01%Triton X100 in 25 mM HEPES, pH 7.4. An aliquot of 2 μl of the dilutedsample was added to a spot of an adsorbent array anionic site. Afterincubation at room temperature for 30 min, the remaining sample on theanionic site was transferred to a spot of adsorbent array normal phasesite. The spot of anionic site was washed with 2 μl of 0.01% Triton X10025 mM HEPES two times. Each wash was accomplished by pipetting the washsolution in and out of the spot ten times. The washes were combined withthe sample initially added on the normal phase spot.

[0586] After incubation at room temperature for 30 min, the remainingsample on the normal phase site was transferred to a spot of adsorbentarray Ni(II) site. The spot of normal phase site was washed with 2 μl ofphosphate buffered saline two times. Each wash was accomplished bypipetting the wash solution in and out of the spot ten times. The washeswere combined with the sample initially added on the Ni(II) spot.

[0587] After the sample was concentrated to near dryness on the Ni(II)spot, the unbound analytes were recovered by washing with 2 μl of 100 mMimidazole in phosphate buffered saline two times. Each wash wasaccomplished by pipetting the wash solution in and out of the spot tentimes. The washes were transferred to a spot of adsorbent arrayaliphatic hydrophobic site.

[0588] The sample was allowed to concentrate to near dryness on thehydrophobic site, unbound analytes were removed by washing with 2 μl of5% acetonitrile in 0.1% trifluoroacetic acid two times. Each wash wasaccomplished by pipeting the wash solution in and out of the spot tentimes.

[0589] Each spot of anionic, normal phase, Ni(II), and hydrophobic sitewas washed with 2 μl of water to remove remaining buffer. An aliquot of0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5%trifluoroacetic acid was added to each spot. The retained analytes oneach site was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

[0590] FIGS. 19A-19D show the retention map of Hemophilus lysate onadsorbent array. Multiple peaks in the mass range 3000 to 25000 Da wereobserved on the adsorbents. Note that each adsorbent shows differentretention for each of the analytes in the sample.

[0591] VIII. Progressive Resolution of an Analyte

[0592] By adding new binding or elution characteristics to a selectivitycondition that resolves an analyte, one can develop a selectivitycondition that provides improved resolution of the analyte. In thisexample, a sample was bound to a Cu(II) adsorbent and exposed to a firsteluant and two second eluants. The second eluants differed from thefirst by the addition of another elution condition. Each added conditionimproved resolution of the analyte.

[0593] Hemophilus wild type stationary phase lysate prepared in 10%glycerol was diluted 1:1 in 20 mM sodium phosphate, 0.5 M sodiumchloride, pH 7.0. After centrifugation, an aliquot of 150 μl of thesupernate was incubated with each spot of adsorbent array Cu(II) site ina bioprocessor. After mixing in the cold for 30 min, the sample wasremoved. Each spot was washed with a different lysate. A first spot waswashed with 150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH7.0. A second spot was washed with 150 μl of 0.05% Triton X100 inaddition to 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0. A third spotwas washed with 150 μl of 100 mM imidazole in addition to 20 mM sodiumphosphate, 0.15 M NaCl, pH 7.0. Each wash was accomplished by incubatingthe wash solution with the spot for 5 min with mixing. The wash wasrepeated two times. Each spot was washed with water to remove detergentand buffer.

[0594] The adsorbent array was removed from the bioprocessor. An aliquotof 0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5%trifluoroacetic acid was added to each spot. The retained analytes oneach spot was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

[0595] FIGS. 20A-20C show the retention map of Hemophilus lysate onadsorbent array Cu(II) site after washing under the three elutionconditions described above. Multiple peaks in the mass range 2000 to18000 Da were observed. The protein marked with a “*” was only a minorcomponent in the retention map of FIG. 20A. When the selectivitycondition was modified by the addition of a detergent, Triton X100 (FIG.20B), to the same buffer, the same protein “*” was retained better thanthe other analytes and resolved better. When the selectivity conditionwas modified by the addition of an affinity displacer, imidazole, to thesame buffer (FIG. 20C.), the protein “*” was highly resolved from theother analytes in the retentate map.

[0596] This strategy of progressive identification of selectivityconditions with improved resolution for an analyte can be adopted todevelop a method for the preparative purification of this protein fromthe total Hemophilus lysate.

[0597] IX. Differential Expression of an Analyte: Marker ProteinDiscovery.

[0598] A. Human Serum

[0599] An aliquot of 0.5 μl of normal or diseased human sera was dilutedwith an equal volume of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0. Eachwas applied to a different spot on an adsorbent array Cu(II) site. Afterincubation at 4° C. for 1 h, each spot was washed with 2 μl of 20 mMsodium phosphate, 0.5 M NaCl, pH 7.0, two times. Each wash wasaccomplished by pipeting the wash solution in and out of the spot tentimes. Each spot was finally washed with 2 μl of water to removeremaining buffer. An aliquot of 0.3 μl of sinapinic acid solution in 50%acetonitrile 0.5% trifluoroacetic acid was added to each spot. Theretained analytes on each spot was analyzed with laserdesorption/ionization time-of-flight mass spectrometer.

[0600] Proteins marked with a “*” in FIG. 21D are present insignificantly greater amounts in diseased serum than in normal serum.The results illustrate a method for discovery of disease markers thatcan be used in clinical diagnostics.

[0601] B. Mouse Urine

[0602] An aliquot of 1 μl of normal, diseased or drug treated mouseurine was applied to a different spot of an adsorbent array Cu(II) site.After incubation at room temperature for 10 min, each spot was washedwith 2 μl of 100 mM imidazole in 20 mM sodium phosphate, 0.15 M NaCl, pH7.0, two times. Each wash was accomplished by pipeting the wash solutionin and out of the spot ten times. Each spot was finally washed with 2 μlof water to remove remaining buffer. An aliquot of 0.3 μl of sinapinicacid solution in 50% acetonitrile 0.5% trifluoroacetic acid was added toeach spot. The retained analytes on each spot was analyzed with laserdesorption/ionization time-of-flight mass spectrometer.

[0603] The retentate maps of normal (control), diseased and drug treatedmouse urine are shown in the FIG. 1. One analyte was found to be presentin much higher quantity in the disease mouse urine (middle panel), thesame analyte was not found in normal mouse urine (upper panel), andfound in drug treated mouse urine in much reduced quantity (lowerpanel). This analyte can be used as a potential disease marker. Toillustrate the feasibility of a quantitative diagnostic assay, the areaunder the peak of the retained marker protein are calculated and shownin the table. A clear quantitative difference is observed between thedisease and drug treated mouse urines. To compensate for experimentalvariability, an internal standard analyte was used. The normalizeddisease marker peak area (i.e., peak area of marker divided by peak areaof internal standard) for each urine sample is presented in the bottompanel. There is at least a ten fold reduction of the disease urinemarker after drug treatment.

[0604] C. Human Urine

[0605] Urines from normal human and cancer patients were diluted 1:2 in0.01% Triton X100 in phosphate buffered saline. An aliquot of 1.5 μl ofnormal or disease human urine was applied to a different spot of anadsorbent array aliphatic hydrophobic site prewetted with 0.5 μl ofisopropanol/acetonitrile (1:2) 0.1% trifluoroacetic acid. Afterincubation at 4 C for 30 min, each spot was washed with 2 μl of 50%ethylene glycol in 10 mM TrisHCl, 0.05 M NaCl, pH 7.5, two times. Eachwash was accomplished by pipeting the wash solution in and out of thespot ten times. Each spot was finally washed with 2 μl of water toremove remaining ethylene glycol and buffer. An aliquot of 0.3 μl ofsinapinic acid solution in 50% acetonitrile 0.5% trifluoroacetic acidwas added to each spot. The retained analytes on each spot was analyzedwith laser desorption/ionization time-of-flight mass spectrometer.

[0606] The retentate maps of urines of four cancer patients and a normalhuman are shown FIG. 23A. Multiple protein peaks were retained on theadsorbent array hydrophobic site after washing with 50% ethylene glycolin Tris/NaCl buffer. To identify possible disease markers, differencemaps between individual patient urine and normal urine are plotted. Eachbar in the difference plot above the baseline represents an analytepresent in higher quantity in the patient urine. (FIG. 23B-23D.)Variations in the patterns of difference map of the patients reflectindividual fluctuations in a population. However, one analyte around5000 Da (marked with *) and a cluster of analytes around 7500 Da (markedwith *), are found to be consistently present in higher quantities inall patients, therefore these can be identified as potential diseasemarkers.

[0607] X. Capture of Phage from Phage Display Library

[0608] Viruses adsorbed to the surface of a protein chip can be detectedby desorption spectrometry. Antibodies against viral coat proteins, usedas adsorbents, can capture viruses. A target protein used as anadsorbent can capture phage displaying a single-chain antibody againstthe target.

[0609] A. Detection Sensitivity of Phage Display Antibody by AdsorbentSubstrate

[0610] M13 phage (10¹² particle/ml) in growth medium was seriallydiluted into 0.01% Triton X100 in 25 mM HEPES, pH 7.4. An aliquot of0.25 μl of each of the diluted phage suspension was added to a spot ofan adsorbent array aliphatic hydrophobic site. An aliquot of 0.3 μl ofCHCA in 50% acetonitrile, 0.5% trifluoroacetic acid was added. Thesamples were analyzed by laser desorption/ionization time-of-flight massspectrometer.

[0611] The M13 phage Gene VIII protein was detected with highsensitivity on the array. FIGS. 24A-24E. A detectable signal(signal/noise>2) was obtained when the phage suspension was diluted10,000,000 times.

[0612] B. Identification of M13 Phage by Adsorbent Array

[0613] Rabbit anti-M13 antibody (Strategene) was immobilized on ProteinA Hyper D (BioSepra), and washed with phosphate buffered saline, pH 7extensively. An aliquot of 1-10 μl suspension of M13 phage (10¹²particle/ml) in growth medium was incubated with 1 μl aliquot ofimmobilized anti-M13 antibody at 4° C. overnight. After washing with0.05% Tween 20 in phosphate buffered saline, pH 7 and then with water toremove detergent and buffer, an aliquot of the captured phage wasanalyzed with laser desorption/ionization time-of-flight massspectrometer in the presence of sinapinic acid.

[0614] The anti-M13 antibody control shows only the antibody signal(singly and doubly charged). FIG. 25A. When the M13 phage was capturedby the antibody, the most easily identifiable protein peaks from thephage are the Gene VIII protein and the Gene III protein fusion withsingle chain antibody. FIG. 25B. Since the M13 phage Gene VIII proteinis detected with high efficiency by the method, it can be used as asensitive monitor of phage capture.

[0615] C. Specific Capture of M13 Phage Displaying Single Chain Antibody

[0616] HIV-1 Tat protein (McKesson BioServices) was coupled to apreactivated substrate. After blocking with ethanolamine, the array waswashed with 0.005% Tween20 in phosphate buffered saline, pH 7, and then0.1% BSA in phosphate buffered saline, pH 7. A serial dilution of M13phage displaying single chain antibody against the Tat protein wasincubated with the Tat protein adsorbent array at 4° C. overnight. Anegative control of a serial dilution of M13 phage not displaying thesingle chain antibody against the Tat protein was also incubated withthe Tat protein adsorbent array the same way. The arrays were washedwith 0.05% Tween20 in phosphate buffered saline, followed by 1 M urea inphosphate buffered saline, pH 7.0 and finally with water to removebuffer and urea. An aliquot of 0.3 μl of CHCA in 50% acetonitrile 0.5%trifluoroacetic acid was added. The retained phage was analyzed by laserdesorption/ionization time-of-flight mass spectrometer.

[0617] A specific binding of M13 phage displaying single chain antibodyagainst Tat protein was observed in a concentration dependent manner(solid line). FIGS. 26A-26D. Nonspecific binding by a nonspecific M13phage was minimal on the adsorbent array (dashed line). These resultsillustrate a very sensitive method of detecting a phage containing agene that encodes a single chain antibody specifically recognizing atarget analyte.

[0618] XI. Screening to Determine Whether a Compound Inhibits BindingBetween Receptor and Ligand

[0619] The methods of this invention can be used to determine whether atest agent modulates the binding of a ligand for a receptor. In thisexample, we show that retentate chromatography can detect the inhibitionof binding between TGF-β and bound TGF-β receptor used as an adsorbentby free TGF-β receptor.

[0620] TGF-β recombinant receptor-Fc fusion protein (R&D, Minnesota) wasspecifically bound on a Protein G adsorbent array. TGF-β (R&D,Minnesota) was serially diluted into cell conditioned medium(2.5×concentrated) and incubated with the receptor-Fc Protein Gadsorbent array at 4° C. overnight. Another set of serially dilutedTGF-β in cell conditioned medium was incubated with the receptor-FcProtein G adsorbent array in the presence of a modulating agent. In thisillustration, the modulating agent was the free TGF-β receptor. Afterincubation under the same conditions, the chips were washed with 0.05%Triton X100 in PBS and then 3M urea in PBS. An aliquot of 0.3 μl ofsinapinic acid was added to each spot and analyzed by laserdesorption/ionization time-of-flight mass spectrometry.

[0621]FIG. 27A shows the specific binding of 1 μg/ml TGF-β to thereceptor-Fc Protein G adsorbent array (solid line). Little or noproteins in the cell conditioned medium were found to bind. FIG. 27Bshows the specific binding of 100 ng/ml of TGF-β to the receptor-FcProtein G adsorbent array (solid line). When the incubation of TGF-β andthe receptor-Fc Protein G adsorbent array was performed in the presenceof a modulating agent (free TGF-β receptor), the binding was completelyeliminated when there was 100 ng/ml of TGF-β (FIG. 27A, dashed line) andonly a trace of binding where there was 1 μg/ml of TGF-β (FIG. 27B,dashed line). In this illustration, the modulating agent (the samereceptor) has high specific binding affinity for the ligand, thusoffering a very effective competition of the target analyte bindingevent. In the other cases, the ratio of the target analyte bound to theadsorbent in the present and absence of the modulating agent gives anindication of the efficacy of the modulating agent.

[0622] XII. Resolving Power of Retentate Chromatography

[0623] This example demonstrates the ability of retentatechromatography, with its parallel processing of a sample under differentselectivity conditions, to resolve proteins in a sample.

[0624] Hemophilus influenzae lysate was prepared in 10% glycerol. Aftercentrifugation, the supernate was diluted 1:3 in 0.01% Triton X100 in 25mM HEPES, pH 7.4. An aliquot of 2 μl of the diluted sample was added toa spot of adsorbent array cationic site. After incubation at roomtemperature for 30 min, the spot was washed with 25 mM HEPES, pH 7.4. Asecond aliquot of 2 μl of the dilute sample as added to a spot ofadsorbent array aliphatic hydrophobic site. After incubation at roomtemperature for 30 min, the spot was washed with water. A third aliquotof 2 μl of the diluted sample was added to a spot of adsorbent arrayCu(II) site. After incubation at room temperature for 30 min, the spotwas washed with 0.05% Triton X100 in phosphate buffered saline, pH 7.4.An aliquot of 0.3 of sinapinic acid solution in 50% acetonitrile 0.5%trifluoroacetic acid was added to each spot. The retained analytes oneach site was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

[0625] Results are shown in FIGS. 28-31. The total retained analytecount was around 550. The result illustrates a method for combinatorialseparation, including separation and detection of multiple analytes inparallel.

[0626] XIII. Sequential Assembly of Multimeric Structures

[0627] This example illustrates a method of building a secondaryadsorbent on a primary adsorbent. The secondary adsorbent then acts as aspecific adsorbent for a target analyte.

[0628] An aliquot of 0.5 μl of GST fusion receptor diluted in 20 mM Tris100 mM, sodium chloride. 0.4% NP40, pH 7.2, was added to a spot of anadsorbent array normal site. The solution was allowed to concentrate onthe spot until almost dryness. The spot was washed with 2 μl of 10 mMTris, 50 mM sodium chloride, pH 7.2, three times. Each wash wasaccomplished by pipeting the wash solution in and out of the spot fivetimes. The spot was finally washed with 2 μl of water two times toremove remaining buffer. An aliquot of 0.3 μl of sinapinic acid solutionin 50% acetonitrile, 0.5% trifluoroacetic acid was added to the spot.The retained GST fusion receptor was analyzed with laserdesorption/ionization time-of-flight mass spectrometer. (FIG. 32.)

[0629] An aliquot of 0.5 μl of GST fusion receptor in 20 mM Tris, 100 mMsodium chloride, 0.4% NP40, pH 7.2, was added to a spot of an adsorbentarray normal site. A sample containing only GST protein (with noreceptor) was applied to another spot as a negative control. Thesolution was allowed to concentrate on the spot until almost dryness.0.5 μl of 10 mM Tris, 50 mM sodium chloride, pH 7.2, was added to eachspot. The solution was removed using a pipet after 10 seconds ofstanding at room temperature.

[0630] An aliquot of 1 μl of a solution containing one specific ligandin a library of 96 other ligands was immediately added to each spot. Theadsorbent array was incubated in a moist chamber at room temperature for1 hour. Each spot was washed with 2 μl of 30% isopropanol:acetonitrile(1:2) in water, two times. Each wash was accomplished by pipeting thewash solution in and out of the spot ten times. An aliquot of 0.3 μl ofα-cyano-4-hydroxycinnamic acid solution in 50% acetonitrile, 0.5%trifluoroacetic acid was added to the spot. The captured ligand on thereceptor was analyzed with laser desorption/ionization time-of-flightmass spectrometer.

[0631]FIG. 33A shows the binding of a specific ligand out of a libraryof 96 other ligands to the GST fusion receptor which is captured on anadsorbent array normal site. FIG. 33B shows that there is no binding ofthe ligand to GST protein alone (with no receptor) captured on the samearray, which serves as a negative control of the experiment.

[0632] The present invention provides novel materials and methods forretentate chromatography. While specific examples have been provided,the above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

[0633] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument Applicants do not admit that any particular reference is “priorart” to their invention.

What is claimed is:
 1. A method for identifying a ligand for a receptorcomprising the steps of: a) providing a substrate comprising anadsorbent wherein the receptor is bound to the adsorbent; b) exposingthe bound receptor to a sample containing the ligand under conditions toallow binding between the receptor and the ligand; and c) detectingbound ligand by desorption spectrometry.
 2. A method of detecting agenetic package containing a polynucleotide that encodes a polypeptideagent that specifically binds to a target adsorbent, the methodcomprising the steps of: a) providing a substrate comprising a targetadsorbent; b) providing a display library that comprises a plurality ofdifferent genetic packages, each different genetic package comprising apolynucleotide that comprises a nucleotide sequence that encodes apolypeptide agent, and each different genetic package having a surfaceon which the encoded polypeptide agent is displayed; c) exposing thesubstrate to the display library under elution conditions to allowspecific binding between a polypeptide agent and the target adsorbent,whereby a genetic package comprising the polypeptide agent is retainedon the substrate; and d) detecting a genetic package retained on thesubstrate by desorption spectrometry.
 3. The method of claim 2 whereinthe display library is a phage display library.
 4. The method of claim 2wherein the step of providing the substrate comprising the targetadsorbent comprises the steps of: i) providing a substrate comprising anadsorbent, wherein the adsorbent retains a target analyte under anelution condition; and ii) exposing the adsorbent to the target analyteunder the elution condition to allow retention of the target analyte bythe adsorbent, whereby the target analyte becomes the target adsorbent.5. The method of claim 2 further comprising the step of (e) sequencingthe nucleotide sequence that encodes the polypeptide agent.
 6. Themethod of claim 2 further comprising the step of (e) isolating theretained genetic package.
 7. The method of claim 2 further comprisingthe step of (e) producing the polypeptide agent.
 8. The method of claim2 wherein the substrate comprises (1) an adsorbent that binds ananchoring polypeptide and (2) at least one target genetic package havinga surface displaying the anchoring polypeptide and a target adsorbentpolypeptide, the target genetic package comprising a polynucleotide thatcomprises a nucleotide sequence that encodes the target adsorbent,wherein the target genetic package is bound to the adsorbent through theanchoring polypeptide.
 9. The method of claim 2 wherein the substratecomprises a cell or cell membrane.
 10. The method of claim 2 wherein thetarget adsorbent comprises a polypeptide that is differentiallyexpressed between cells of different phenotypes.
 11. The method of claim3 wherein the phage is M13.
 12. The method of claim 4 wherein the targetanalyte is a target polypeptide and the step of ii) exposing theadsorbent comprises the step of producing the target polypeptide in situon the adsorbent by in vitro translation of a polynucleotide encodingthe target polypeptide.
 13. The method of claim 5 wherein the step ofsequencing comprises amplifying the polynucleotide sequence in situ onthe substrate.
 14. The method of claim 7 wherein the step of producingcomprises reproducing the retained genetic package that displays thepolypeptide agent.
 15. The method of claim 7 comprising expressing thepolypeptide agent from an expression vector that comprises an expressioncontrol sequence operatively linked to the nucleotide sequence encodingthe polypeptide agent.
 16. The method of claim 7 further comprising thestep of producing a substrate comprising an adsorbent that comprises thepolypeptide agent.
 17. The method of claim 8 wherein the at least onetarget genetic package is selected from a target display libraryscreened for genetic packages that bind at least one primary targetanalyte and wherein the adsorbent comprises the primary target analyte.18. The method of claim 11 wherein the polypeptide agent is a singlechain antibody.
 19. The method of claim 12 wherein the targetpolypeptide is produced in situ by in vitro translation of apolynucleotide encoding the target polypeptide.
 20. The method of claim14 wherein the step of reproducing is carried out in situ on thesubstrate.
 21. The method of claim 19 wherein the polynucleotideencoding the target polypeptide is produced in situ by in vitrotranscription.
 22. A substrate for desorption spectrometry comprising anadsorbent that binds an anchoring polypeptide displayed on a surface ofa genetic package, wherein the surface of the genetic package furtherdisplays a target polypeptide and wherein the genetic package comprisesa polynucleotide comprising a nucleotide sequence that encodes thetarget polypeptide.
 23. The substrate of claim 22 wherein the geneticpackage is an M13 phage.
 24. The substrate of claim 22 wherein theanchoring polypeptide is a fusion polypeptide with gene III protein andthe target polypeptide is a fusion polypeptide with gene VIII protein.25. A substrate comprising an adsorbent that comprises a polypeptideagent that specifically binds to a target analyte, the polypeptide agentidentified by the method of claim
 33. 26. The substrate of claim 25wherein the polypeptide agent is a single chain antibody.
 27. A methodfor detecting translation of a polynucleotide comprising the steps of:a) providing a substrate comprising an adsorbent for use in desorptionspectrometry; b) contacting the substrate with the polynucleotideencoding a polypeptide and with agents for in vitro translation of thepolynucleotide, whereby the polypeptide is produced; c) exposing thesubstrate to an eluant to allow retention of the polypeptide by theadsorbent; and d) detecting retained polypeptide by desorptionspectrometry; whereby detection of the polypeptide provides detection oftranslation of the polynucleotide.
 28. A method comprising the steps of:a) exposing a first sample to a primary adsorbent and to an eluant toallow retention of a first analyte by the adsorbent, and detecting theadsorbed analyte by desorption spectrometry, whereby the retained firstanalyte becomes a secondary adsorbent; b) exposing a second sample tothe secondary adsorbent and to an eluant to allow retention of a secondanalyte by the secondary adsorbent, and detecting the adsorbed secondanalyte by desorption spectrometry, whereby the retained second analytebecomes a tertiary adsorbent.
 29. The method of claim 28 furthercomprising repeating step (b) at least once for a subsequent sample orsamples.
 30. A screening method for determining whether an agentmodulates binding between a target analyte and an adsorbent comprisingthe steps of: a) providing a substrate comprising an adsorbent to whichthe target analyte binds under an elution condition; b) exposing thesubstrate to the target analyte and to the agent under the elutioncondition to allow binding between the target analyte and the adsorbent;c) detecting an amount of binding between the target analyte and theadsorbent by desorption spectrometry; and d) determining whether themeasured amount is different than a control amount of binding when thesubstrate is exposed to the target analyte under the elution conditionwithout the agent; whereby a difference between the measured amount andthe control amount indicates that the agent modulates binding.
 31. Themethod of claim 30 wherein the adsorbent comprises a ligand thatspecifically binds the target analyte.
 32. The method of claim 30wherein the adsorbent comprises a genetic package having a surface thatdisplays a polypeptide ligand that specifically binds the targetanalyte.
 33. The method of claim 30 for screening a combinatoriallibrary of agents comprising exposing each of a plurality of agents inthe library to each of a plurality of the adsorbents.
 34. The method ofclaim 31 wherein the ligand is an enzyme and the target analyte is asubstrate of, or an inhibitor for, the enzyme, or vice-versa.
 35. Themethod of claim 31 wherein the ligand is a hormone and the targetanalyte is a cell surface receptor or an intracellular receptor of thehormone, or vice-versa.